Chromophore-based methods and kits for detecting genetic variations in polynucleotide analytes

ABSTRACT

Methods and kits for detecting a genetic variation in a polynucleotide analyte in a sample. A fluorophore is attached to a first primer, a quencher is attached to a second primer, and the first primer and the second primer are specific for the polynucleotide analyte. At least one of the primers is configured to hybridize to a region of the polynucleotide analyte encoding the genetic variation. The primers are configured to amplify the polynucleotide analyte having the genetic variation and a corresponding polynucleotide analyte lacking the generic variation. There is a detectable difference between a change in signal generated by the fluorophore and quencher when using the first and second primers to amplify the polynucleotide analyte with the genetic variation, and a change in signal generated by the fluorophore and quencher when using the first and second primers to amplify the corresponding polynucleotide analyte lacking the genetic variation.

CROSS-REFERENCE

This application is a continuation application of U.S. patentapplication Ser. No. 15/675,048 filed on Aug. 11, 2017, which issued asU.S. Pat. No. 10,081,844 on Sep. 25, 2018, which is a continuationapplication of U.S. patent application Ser. No. 14/162,725 filed on Jan.23, 2014, now abandoned, which claims the benefit of U.S. provisionalapplication 61/756,343, filed Jan. 24, 2013, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 6, 2017, isnamed 38075-719_301_SL.txt and is 12,793 bytes in size.

BACKGROUND OF THE INVENTION

Nucleic acid analyte identification is a critical procedure in a varietyof biomedical applications, such as in research and clinical diagnosticenvironments. Identification of an analyte is primarily done bysequencing or by amplification-based detection. For example, in thelatter scheme, the polymerase chain reaction is often used to increasethe quantity of the nucleic acid analyte present. Then, the nucleic acidanalytes are discriminated using one of several additional techniquesincluding fluorescence intensity measurement (e.g., fluorescent probesor intercalating dyes), length discrimination (e.g., using gelelectrophoresis or melt curve analysis), or chromatography (e.g.,haptin-based nucleic acid capture). Thus, current amplification-baseddetection technology indirectly detects analytes and requires asecondary technique (such as gel electrophoresis or mass spectroscopy)for analyte detection. Amplification or polymerization-based techniquesthat directly detect analytes would improve efficiency, time and cost.

SUMMARY OF THE INVENTION

Disclosed herein are methods, compositions, and kits for detectinganalytes, particularly polynucleotides and/or polypeptides. The methodsgenerally involve using oligonucleotides (e.g., primers, probes)attached to chromophores (e.g., fluorophores, quenchers, etc.) inamplification or polymerization reactions in order to detect apolynucleotide analyte. In some embodiments, provided herein are methodsof detecting at least one polynucleotide analyte in a sample,comprising: (a) combining the sample with a first primer and a firstoligonucleotide, wherein a first chromophore is attached to the firstprimer, a second chromophore is attached to the first oligonucleotide,the first primer and the first oligonucleotide are specific for a firstpolynucleotide analyte and the first chromophore is different from thesecond chromophore; (b) measuring a first signal generated by the firstand second chromophores; (c) performing at least one polymerizationreaction with the first primer using the first polynucleotide analyte asa template; and (d) measuring a second signal generated by the first andsecond chromophores; wherein the first and second signals are used todetect the first polynucleotide analyte.

In some cases, the first oligonucleotide is a second primer. In somecases, the first chromophore is attached to the 5′ end of the firstprimer. In some cases, the first chromophore is an inorganic or organicdye, a fluorophore or a quencher. In some cases, the first chromophoreis a fluorophore. In some cases, the fluorophore is 6-FAM (Fluorescein),6-FAM (NHS Ester), Fluorescein dT, HEX, JOE (NHS Ester), MAX, TET, ROX,TAMRA, TARMA (NHS Ester), TEX 615, ATTO 488, ATTO 532, ATTO 550, ATTO565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665 or TYE705. In some cases, the second chromophore is attached to the 5′ end ofthe second primer. In some cases, the second chromophore is an inorganicor organic dye, a fluorophore or a quencher. In some cases, the secondchromophore is a quencher. In some cases, the quencher is Iowa Black FG,Iowa Black RG, BHQ1, BHQ2 or BHQ3. In some cases, the first chromophoreis a fluorophore and the second chromophore is a quencher.

In some cases, the methods described herein further comprising comparingthe first and second signals, wherein a change in the first and secondsignals indicates the presence of the first polynucleotide analyte. Insome cases, the change in the first and second signals is a decrease influorescent intensity. In some cases, the change is an increase inintensity. In some cases, the decrease in fluorescent intensity is atleast about a 30% decrease in signal (or at least about 10%, 20%, 30%,35%, 40%, 45%, 50%, 55%, 60%, or 75%). In some cases, the second signalis measured after a second polymerization reaction. In some cases, thepolymerization reaction is a polymerase chain reaction process or anisothermal process. In some cases, the polymerase chain reaction processis an end-point polymerase chain reaction process, a real-timepolymerase chain reaction process, a digital polymerase chain reactionprocess, a droplet digital polymerase chain reaction process, or aquantitative polymerase chain reaction process. In some cases, the firstprimer is a forward primer and the second primer is a reverse primer. Insome cases, the first primer is a reverse primer and the second primeris a forward primer. In some cases, the first and second chromophoresinteract through an electron-transfer process. In some cases, the firstpolynucleotide analyte is from about 10 to about 500 nucleotides inlength. In some cases, the concentration of the first polynucleotideanalyte is from about 10 μM to about 10 aM. In some cases, the firstpolynucleotide analyte is a DNA polynucleotide analyte. In some cases,the first polynucleotide analyte is an RNA polynucleotide analyte.

In some cases, the first polynucleotide analyte comprises a geneticvariation. In some cases, the genetic variation comprises asubstitution, an addition, a deletion or a translocation. In some cases,the genetic variation comprises a single-nucleotide polymorphism (SNP).In some cases, the at least one polynucleotide analyte is from a sourceselected from a human, a non-human mammal, a plant, a bacteria, afungus, an archaea, a parasite, or a virus. In some cases, the virus isa human immunodeficiency virus, an influenza type A virus, an influenzatype B virus, a respiratory syncytial virus type A (RsvA), a respiratorysyncytial virus type B virus (RsvB), a human rhinovirus (Hrv), a humanmetapneumovirus (Hmpv) or a human parainfluenza virus type 3 (PIV-3). Insome cases, the sample is a forensic sample, a clinical sample, a foodsample, an environmental sample, a pharmaceutical sample, or a samplefrom a consumer product. In some cases, the methods disclosed hereinfurther comprising detecting at least one additional polynucleotideanalyte within the sample with an additional fluorophore attached to aprimer, wherein the first chromophore is a fluorophore with the samecolor as the additional fluorophore. In some cases, the first primer isnot attached to two or more chromophores. In some cases, the firstoligonucleotide is not attached to two or more chromophores. In somecases, step b) of the method further comprises measuring the firstsignal at a first denaturing step and a third signal at a firstannealing step; step d) further comprises measuring the second signal ata second denaturing step and a fourth signal at a second annealing step;and the method further comprises comparing the first signal with thesecond signal to obtain a first ratio and comparing the third signalwith the fourth signal to obtain a second ratio; wherein when the firstratio is about 1 and the second ratio is greater than or less than 1indicate the presence of the first polynucleotide analyte.

In some cases, step a) of the method described herein further comprisescombining the sample with a third primer and a second oligonucleotide,wherein a third chromophore is attached to the third primer and a fourthchromophore is attached to the second oligonucleotide, and the thirdprimer and the second oligonucleotide are specific for a secondpolynucleotide analyte; step b) further comprises measuring a fifthsignal generated by the third and fourth chromophores; step d) furthercomprises measuring a sixth signal generated by the third and fourthchromophores; wherein the fifth and sixth signals are used to detect thesecond polynucleotide analyte. In some cases, the methods disclosedherein further comprising comparing the fifth and sixth signals, whereina change in the fifth and sixth signals indicates the presence of thesecond polynucleotide analyte. In some cases, the second oligonucleotideis a fourth primer.

Also disclosed herein are methods of detecting at least one geneticvariation in an analyte comprising: (a) combining a first analyte with afirst primer and a second primer, wherein a first chromophore isattached to the first primer, a second chromophore is attached to thesecond primer, at least one of the first and the second primers arespecific for a first genetic variation in the first analyte and thefirst chromophore is different from the second chromophore; (b)measuring a first signal generated by the first and second chromophores;(c) performing at least one polymerization reaction with the firstprimer and the second primer using the first analyte as a template; and(d) measuring a second signal generated by the first and secondchromophores; wherein the first and second signals are used to detectthe first genetic variation in the first analyte. In some cases, themethods described herein further comprise comparing the first and secondsignals, wherein a change in the first and second signals indicates thepresence of the genetic variation in the first analyte.

In some cases, the analyte is a polynucleotide analyte. In some cases,the genetic variation comprises a substitution, an addition, a deletionor a translocation. In some cases, the genetic variation comprises asingle-nucleotide polymorphism (SNP). In some cases, the first primercomprises a sequence encoding the SNP or the first primer binds to aregion of the analyte encoding the SNP. In some cases, the second primercomprises a sequence not encoding the SNP or the second primer comprisesa sequence complementary to a region of the analyte not encoding theSNP. In some cases, the first primer encodes a region of the analyteless than 500 base pairs apart from a region of the analyte encoded bythe second primer. In some cases, the change in signal is distinct forat least two of the mismatched base pairs selected from the groupconsisting of UU, UT, UG, UC, UA, AA, TT, GG, CC, AG, AC, TG and TC. Insome cases, the change in signal from a mismatched base pair is distinctfrom a change in signal from a complementary base pair.

In some cases, step a) of the methods described herein further comprisescombining the first analyte with a third primer and a fourth primer,wherein a third chromophore is attached to the third primer, a fourthchromophore is attached to the fourth primer, the third and the fourthprimers are specific for a second genetic variation in the first analyteand the third chromophore is different from the fourth chromophore; stepb) further comprises measuring a third signal; step d) further comprisesmeasuring a fourth signal; and the method further comprises comparingthe third and fourth signals; wherein a change in the third and fourthsignals indicates the presence of the second single genetic variation inthe first analyte. In some cases, step a) of the method furthercomprises combining a second analyte with a third primer and a fourthprimer, wherein a third chromophore is attached to the third primer, afourth chromophore is attached to the fourth primer comprises, the thirdand the fourth primers are specific for a second genetic variation inthe second analyte and the third chromophore is different from thefourth chromophore; step b) further comprises measuring a third signal;step d) further comprises measuring a fourth signal; and the methodfurther comprises comparing the third and fourth signals; wherein achange in the third and fourth signals indicates the presence of thesecond single genetic variation in the second analyte. In some cases,step a) further comprises combining a second analyte with a third primerand a fourth primer, wherein the first chromophore is attached to thethird primer, the second chromophore is attached to the fourth primer,and the third and the fourth primers are specific for a second geneticvariation in the second analyte. In some cases, the polymerizationreaction is a PCR process or an isothermal reaction. In some cases, thePCR process is an end-point PCR process, a digital PCR process, areal-time PCR process, a droplet digital PCR process, or a quantitativePCR process. In some cases, the polymerization reaction is aquantitative PCR process. In some cases, the detecting comprises aquantitative PCR method. In some cases, the detecting comprises aquantitative PCR method and a second method. In some cases, the secondmethod is a digital PCR process. In some cases, at least one SNP isdetected in a gene. In some cases, at least one SNP is associated with adisease. In some cases, the disease is a genetic disorder, an autoimmunedisease, a neurological disease, a cardiovascular disease, or a cancer.

Also disclosed herein are methods of detecting a plurality of analytes,comprising: a) combining the plurality of analytes with a plurality ofprimer pairs, wherein each primer pair is specific to a single analyteand each primer of the primer pair is attached to at least onechromophore; b) measuring a first set of signals generated by thechromophores; c) performing at least one polymerization reaction withthe plurality of primer pairs using the plurality of analytes astemplates; and d) measuring a second set of signals generated by thechromophores; wherein the first and second set of signals are used todetect each analyte of the plurality of analytes. In some cases, themethod described herein further comprises: e) repeating step c and d atleast once; and f) generating a set of signature profiles; wherein thepresence of each analyte of the plurality of analytes is detected bycomparing the set of signature profiles to a control set of signatureprofiles.

In some cases, the signature profile is an end-point signature profileor a signature curve. In some cases, the plurality of analytes hasdifferent lengths. In some cases, each of the plurality of primer pairscomprises a forward primer and a reverse primer. In some cases, one, twoor more chromophores are attached to the forward primer. In some cases,one chromophore is attached to the 5′ end of the forward primer. In somecases, at least one chromophore is attached to the reverse primer. Insome cases, one chromophore is attached to the 5′ end of the reverseprimer. In some cases, the chromophore is an inorganic or organic dye, afluorophore or a quencher. In some cases, the chromophore is afluorophore. In some cases, the chromophore is a quencher.

Also disclosed herein are methods of generating a signature curveprofile for a polynucleotide analyte, comprising: (a) contacting thepolynucleotide analyte with a first primer and a second primer, whereina first chromophore is attached to the first primer and a secondchromophore is attached to the second primer, the first primer and thesecond primer are specific for the polynucleotide analyte and the firstchromophore is different from the second chromophore; (b) measuring afirst signal at a first temperature; (c) performing at least onepolymerization reaction with the first primer and the second primerusing the polynucleotide analyte as a template; (d) measuring a secondsignal at the first temperature; and (e) repeating step c and d at leastonce; wherein the signals create the signature curve profile of thepolynucleotide analyte. In some cases, the method disclosed hereinfurther comprises: f) changing the temperature; g) measuring a thirdsignal at a second temperature; and h) repeating steps f and g at leastonce; wherein the signals create the signature curve profile of thepolynucleotide analyte.

In some cases, the signature curve is a length curve, a morphologycurve, a melt curve, or a SNP curve. In some cases, the polymerizationreaction is a polymerase chain reaction process. In some cases, thepolymerase chain reaction process is an end-point polymerase chainreaction process, a real-time polymerase chain reaction process, adigital polymerase chain reaction process or a quantitative polymerasechain reaction process. In some cases, the first chromophore is aninorganic or organic dye, a fluorophore or a quencher. In some cases,the first chromophore is a fluorophore. In some cases, the fluorophoreis 6-FAM (Fluorescein), 6-FAM (NHS Ester), Fluorescein dT, HEX, JOE (NHSEster), MAX, TET, ROX, TAMRA, TARMA (NHS Ester), TEX 615, ATTO 488, ATTO532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE563, TYE 665 or TYE 705. In some cases, the second chromophore is aninorganic or organic dye, a fluorophore or a quencher. In some cases,the second chromophore is a quencher. In some cases, the quencher isIowa Black FG, Iowa Black RG, BHQ1, BHQ2 or BHQ3. In some cases, thepolynucleotide analyte is a DNA polynucleotide analyte. In some cases,the polynucleotide analyte is an RNA polynucleotide analyte. In somecases, the polynucleotide analyte is from a source selected from thegroup consisting of a human, a non-human mammal, a plant, a bacteria, anarchaea, a fungus, a parasite, and a virus. In some cases, the virus isa human immunodeficiency virus, an influenza type A virus, an influenzatype B virus, a respiratory syncytial virus type A (RsvA), a respiratorysyncytial virus type B virus (RsvB), a human rhinovirus (Hrv), a humanmetapneumovirus (Hmpv) or a human parainfluenza virus type 3 (PIV-3).

Also disclosed herein is a method of monitoring an amplificationreaction comprising: (a) contacting a first polynucleotide analyte with:i) a first primer and a second primer, wherein the first primer and thesecond primer are specific for the first polynucleotide analyte; and ii)at least two chromophores capable of specific incorporation into aproduct amplified from the first polynucleotide analyte; (b) subjectingthe combination in step (a) to a temperature capable of denaturingdouble-stranded DNA; (c) measuring a first signal in step (b) generatedby the at least two chromophores; (d) subjecting the combination in stepb) to a temperature capable of annealing polynucleotides; (e) measuringa second signal in step (d) generated by the at least two chromophores;and (f) repeating steps b)-e) to obtain a third signal and a fourthsignal; and (g) comparing the first and the third signals to obtain afirst ratio and the second and the fourth signals to obtain a secondratio; wherein an amplification reaction occurs when the first ratio isabout 1 and the second ratio is greater than or less than 1. In somecases, at least one chromophore is attached to the first primer, forexample exactly one chromophore may be attached to the first primer. Insome cases, one chromophore (or exactly one chromophore) is attached tothe 5′ end of the first primer. In some cases, one, two or morechromophores are attached to the second primer. In some cases, onechromophore is attached to the 5′ end of the second primer. In somecases, the chromophore is an inorganic or organic dye, a fluorophore ora quencher.

Also disclosed herein are methods of detecting a morphology of ananalyte, comprising: (a) providing a sample comprising the analyte,wherein a first chromophore, a second chromophore and a thirdchromophore are attached to the analyte, wherein the first, second andthird chromophores are different; (b) measuring a signal from the firstchromophore, second chromophore and the third chromophore at a first andsecond temperature, wherein the first and second temperatures aredifferent; and (c) using the measured signals to detect the morphologyof the analyte. In some cases, the analyte is a protein, a polypeptide,a lipid or a polynucleotide. In some cases, the analyte is apolynucleotide. In some cases, the polynucleotide is a DNApolynucleotide. In some cases, the polynucleotide is an RNApolynucleotide. In some cases, the first chromophore is an inorganic ororganic dye, a fluorophore or a quencher. In some cases, the firstchromophore is a first fluorophore. In some cases, the first fluorophoreis 6-FAM (Fluorescein), 6-FAM (NHS Ester), Fluorescein dT, HEX, JOE (NHSEster), MAX, TET, ROX, TAMRA, TARMA (NHS Ester), TEX 615, ATTO 488, ATTO532, ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE563, TYE 665 or TYE 705. In some cases, the second chromophore is aninorganic or organic dye, a fluorophore or a quencher. In some cases,the second chromophore is a second fluorophore. In some cases, thesecond fluorophore is 6-FAM (Fluorescein), 6-FAM (NHS Ester),Fluorescein dT, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA, TARMA (NHSEster), TEX 615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTO Rho101,ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665 or TYE 705. In somecases, the third chromophore is an inorganic or organic dye, afluorophore or a quencher. In some cases, the third chromophore is aquencher. In some cases, the quencher is Iowa Black FG, Iowa Black RG,BHQ1, BHQ2 or BHQ3. In some cases, the analyte comprises more than onefluorophore. In some cases, the analyte comprises more than onequencher. In some cases, the polynucleotide comprises a quencher at its3′ end.

Also disclosed herein are methods of detecting a polynucleotide analytecomprising: (a) combining the polynucleotide analyte with at least twochromophores, wherein the at least two chromophores are each attached toa separate polynucleotide that is complementary to a region within thepolynucleotide analyte; (b) performing at least one polymerizationreaction to incorporate the at least two chromophores into products ofthe polymerization reaction; and (c) detecting a fluorescent intensityfrom the at least two chromophores at a first timepoint and a secondtimepoint, wherein the second timepoint is later than the firsttimepoint and wherein a change (particularly a decrease) in fluorescentintensity at the second timepoint relative to the first timepoint isindicative of the presence of the polynucleotide analyte. In some cases,the polymerization reaction is a polymerase chain reaction. In somecases, the first timepoint is after step a) and the second timepoint isafter step b). In some cases, the products of the at least onepolymerization reaction each comprise a first polynucleotide strand anda second polynucleotide strand, wherein the first polynucleotide strandand the second polynucleotide strand are complementary. In some cases,the at least two chromophores are different. In some cases, the at leasttwo chromophores comprise a fluorophore. In some cases, the at least twochromophores comprise a fluorophore and a quencher. In some cases, thefluorophore is incorporated into the first polynucleotide strand and thequencher is incorporated into the second polynucleotide strand. In somecases, the fluorophore is incorporated at the 5′ end of firstpolynucleotide strand and the quencher is incorporated at the 5′ end ofthe second polynucleotide strand.

Also disclosed herein are compositions and kits, particularly fordetecting polynucleotide or polypeptide analytes. In some cases, thekits comprise oligonucleotides (e.g., primers, probes, etc.) attached toa chromophore (e.g., fluorophore, quencher). In some cases, the kitcomprises: (a) a first primer or probe attached to a fluorophore; and(b) a second primer or probe attached to a quencher. In some cases, thekit comprises: (a) a first primer or probe attached to exactly one firstchromophore, wherein the first chromophore is a fluorophore; and (b) asecond primer or probe attached to exactly one second chromophore,wherein the first chromophore is different from the second chromophore.In some cases, the second chromophore is a quencher. In some cases, thesecond chromophore is a fluorophore. In some cases, the first primer orprobe is a primer. In some cases, the second primer or probe is aprimer. In some cases, the first primer or probe comprises anoligonucleotide sequence that is complementary to the sequence of atarget analyte. In some cases, the second primer or probe comprises anoligonucleotide sequence complementary to the sequence of a targetanalyte. In some cases, the fluorophore is 6-FAM (Fluorescein), 6-FAM(NHS Ester), Fluorescein dT, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA,TARMA (NHS Ester), TEX 615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTORho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665 or TYE 705. Insome cases, the quencher is Iowa Black FG, Iowa Black RG, BHQ1, BHQ2 orBHQ3. In some cases, the kit comprises at least three primers, whereineach primer is attached to a different chromophore.

In some cases, the kit comprises: (a) a first primer or probe attachedto a first chromophore, wherein the first chromophore is a fluorophore;and (b) a second primer or probe attached to a second chromophore,wherein the first chromophore is different from the second chromophore.In some cases, the kit comprises: (a) a first primer or probe attachedto exactly one first chromophore; and (b) a second primer or probeattached to exactly one second chromophore, wherein the firstchromophore is different from the second chromophore. In some cases, thekit comprises: (a) a first primer or probe attached to exactly one firstchromophore, wherein the first chromophore is a fluorophore; and (b) asecond primer or probe attached to exactly one second chromophore,wherein the first chromophore is different from the second chromophore.In some cases, the second chromophore is a quencher. In some cases, thesecond chromophore is a fluorophore. In some cases, the first primer orprobe is a primer. In some cases, the second primer or probe is aprimer. In some cases, the first primer or probe comprises anoligonucleotide sequence that is complementary to the sequence of atarget analyte. In some cases, the second primer or probe comprises anoligonucleotide sequence complementary to the sequence of a targetanalyte. In some cases, the fluorophore is 6-FAM (Fluorescein), 6-FAM(NHS Ester), Fluorescein dT, HEX, JOE (NHS Ester), MAX, TET, ROX, TAMRA,TARMA (NHS Ester), TEX 615, ATTO 488, ATTO 532, ATTO 550, ATTO 565, ATTORho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665 or TYE 705. Insome cases, the quencher is Iowa Black FG, Iowa Black RG, BHQ1, BHQ2 orBHQ3. In some cases, the kit comprises at least three primers, whereineach primer is attached to a different chromophore.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the invention are set forth with particularity in theappended claims. A better understanding of the features and advantagesof the present invention will be obtained by reference to the followingdetailed description that sets forth illustrative embodiments, in whichthe principles of the invention are utilized, and the accompanyingdrawings of which:

FIG. 1 exemplifies a mechanism for detection of a nucleic acid analyteusing chromophore-attached primers (a fluorophore-attached forward (FWD)primer and a quencher-attached reverse (or rewind (RWD)) primer) duringa polymerase chain reaction (PCR). FIGS. 1A-1B illustrate thedenaturation of a double-stranded polynucleotide analyte. FIGS. 1C-1Dillustrate the annealing of the chromophore-attached primers to oppositestrands of the analyte and for a polymerase to extend the primers duringa PCR reaction. FIGS. 1E-1F illustrate the formation of adouble-stranded PCR product containing both chromophores (fluorophoreand a quencher) which leads to the generation of a signal (e.g.,quenching of fluorescence).

FIG. 2 exemplifies an electron transfer state. FIG. 2A illustrates anenergy transfer formula. FIG. 2B illustrates a Jablonski diagram showingForster resonance energy transfer with typical timescales indicated.

FIG. 3 illustrates a diagnostic protocol and treatment method for usewith a detection method described herein.

FIG. 4 illustrates a conceptual schematic of an exemplary computerserver to be used for processing a method described herein.

FIG. 5A-FIG. 5D exemplify the detection of HIV TPP analytes at varyingconcentrations using quantitative PCR (qPCR). FIG. 5A illustrates theqPCR detection of 80 bp HIV TPP analytes at 10 nM concentration. FIG. 5Billustrates the qPCR detection of 80 bp HIV TPP analytes at 1 nMconcentration. FIG. 5C illustrates the qPCR detection of 80 bp HIV TPPanalytes at 100 pM concentration. FIG. 5D illustrates the qPCR detectionof 80 bp HIV TPP analytes at 10 pM concentration. All successfuldetection curves exhibit the inverse-sigmoidal characteristic.

FIG. 6A-FIG. 6D illustrate the sensitivity of detection of HIV TPP usingan exemplary detection method. FIG. 6A illustrates the qPCR detection of80 bp HIV TPP analytes at 1 pM concentration. FIG. 6B illustrates theqPCR detection of 80 bp HIV TPP analytes at 100 fM concentration. FIG.6C illustrates the qPCR detection of 80 bp HIV TPP analytes at 10 fMconcentration. FIG. 6D illustrates the qPCR detection of 80 bp HIV TPPanalytes at 1 fM concentration.

FIG. 7A-FIG. 7C show that the extent of signal generation (quenching) isstrongly correlated with the distance between a quencher and afluorophore. Analytes were varied in length from 40 bp to 120 bp for asingle set of primers. FIG. 7A illustrates the extent of quenching usinga 40mer analyte. FIG. 7B illustrates the extent of quenching using a60mer analyte. FIG. 7C illustrates the extent of quenching using a 80meranalyte.

FIG. 8A-FIG. 8B illustrate the effect of a single nucleotidepolymorphism on signal generation. Signal level changes significantlywhen a mis-priming event occurs (FIG. 8A and FIG. 8B).

FIG. 9 exemplifies the binary coding of analytes. Analytes in a 3-plexCY3 assay were coded using binary spaced primer concentrations. A TaqManpositive control sequence was amplified in each reaction to confirm thatthe PCR performed successfully.

FIG. 10A-10E exemplify exemplifies an amplification or polymerizationreaction using a polymerase chain reaction (PCR) to detect an analyte.FIG. 10A illustrates the sequence of the analyte (Analyte 01) to bedetected_(SEQ ID NO: 50). FIG. 10B illustrates a forward (FWD) and areverse (RWD) primer pair specific for Analyte 01 (SEQ ID NOs: 51-52,respectively, in order of appearance). FIG. 10C illustrates theannealing step during the first cycle of the PCR reaction (SEQ ID NOs:50, 52 and 51, respectively, in order of appearance). FIG. 10Dillustrates the annealing step during the second cycle of the PCRreaction (SEQ ID NOs: 50, 52 and 51, respectively, in order ofappearance). FIG. 10E illustrates the extended PCR products after thesecond extension cycle (SEQ ID NO: 50).

FIG. 11 exemplifies how a detection method described herein can be usedin a multiplex reaction to detect quenched signals in three channels.

FIG. 12 exemplifies the incorporation of multiple chromophores into ananalyte in order to determine its morphology (SEQ ID NO: 50).

FIG. 13 exemplifies signal generation as a function of temperature foran analyte containing multiple chromophores. FIG. 13A illustrates themorphology of the oligonucleotide at a first temperature (T₁). Threefluorophores (F₁, F₂ and F₃) are attached to the oligonucleotide and onequencher (Q₁) is attached at the 3′ end of the oligonucleotide. FIG. 13Billustrates the fluorescence signature profile of the oligonucleotide atT₁. At T₁, Q1 is closest to F₃, and a quenching effect is observed (FIG.13B). FIG. 13C illustrates the morphology of the oligonucleotide at asecond temperature (T₂). FIG. 13D illustrates the fluorescence signatureprofile of the oligonucleotide at T₂. At T₂, the oligonucleotide isfully denatured and Q₁ is further away from F₃. As a result, thefluorescence signal of F₃ increased (FIG. 13D).

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are methods, systems, compositions, and kits for thedetection of one or more analytes using chromophore-attachedoligonucleotides (e.g., primers and/or probes) specific for an analyte.For example, the incorporation of chromophores into an amplification orpolymerization product (e.g., through the use of forward and reverseprimers that are attached to different chromophores as illustrated,e.g., in FIG. 1) provides a number of advantages.

For example, methods described herein can provide for the directdetection of one or more analytes in a single reaction (such as anamplification reaction (such as a PCR reaction or PCR reaction process)or a polymerization reaction). In addition, the direct incorporation ofchromophores into the amplification or polymerization product (e.g., theanalyte) allows for more accurate quantification of the analyte and forthe real-time monitoring of the progression of an amplification orpolymerization reaction. For example, once the analyte has beenamplified to incorporate two different chromophores, upon excitation ofone chromophore, an electron may travel through the pi-bond network of aDNA backbone to interact with a second chromophore, thereby generating adetectable signal or change in signal.

Methods described herein are particularly suitable for detecting geneticvariations, such as single nucleotide polymorphisms (SNPs) or otherqualitative information of an analyte. In some cases, a single base-pairmismatch between a chromophore-attached primer and the analyte can bedetected upon amplification or polymerization of the analyte (orpolymerization or extension of the primer). For example, a change insignal may occur if there is a disruption in a contiguousdouble-stranded DNA sequence upon amplification or polymerization of theanalyte (or polymerization or extension of the primer (e.g., the primerspecific for the SNP)) when a SNP is present. A single base pairmisalignment (e.g. internal misalignment (such as a SNP) or terminaloverhangs) can result in significant decrease in signal compared to thesignal generated upon amplification or polymerization of the analyte (orpolymerization or extension of the primer) without a base pairmisalignment due to a disruption in electron transport betweenchromophores incorporated into an analyte containing a base pairmisalignment.

Additionally described herein are methods to normalize signals during anamplification or polymerization reaction, such as a PCR reaction (or PCRreaction process). In some cases, the methods described herein canprovide for a cycle-by-cycle normalization of a PCR reaction or process(e.g. chopper stabilization where individual cycles are reset to areference value). For example, in some cases, when a signal measuredduring the denaturing step of a cycle is within a reference signalrange, the denaturation signal can be used to normalize the signalmeasured during the annealing step of the same cycle. Thereby, thecycle-by-cycle normalization can provide a means to determine thereliability of the PCR reaction and can be used with the detectionmethods described herein to provide particularly accurate detection andmeasurements of analytes.

Also described herein are methods which allow detection of analytespresent in low concentrations. In some cases, the sensitivity of themethods described herein can detect analytes at concentrations of about10 uM to about 1 aM. In some cases, the methods provided herein can becombined with a digital amplification or polymerization process (e.g.droplet digital PCR), to further enhance the detection. In some cases,the methods provided herein can be used to detect analytes that arepresent at a trace concentration in a sample (e.g. a rare SNP).

Further described herein are methods to detect of multiple analytes in asingle reaction or experiment, without the need to resort to additionalexperiments or materials (e.g. reagents). Thereby, the disclosed methodscan reduce or eliminate the associated cost of additional reagents ormaterials and increase time and efficiency.

I. Certain Terminology

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed. In this application,the use of the singular includes the plural unless specifically statedotherwise. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. In thisapplication, the use of “or” means “and/or” unless stated otherwise.Furthermore, use of the term “including” as well as other forms, such as“include,” “includes,” and “included,” is not limiting.

The term “about,” as used herein, generally refers to a range that is15% greater than or less than a stated numerical value within thecontext of the particular usage. For example, “about 10” would include arange from 8.5 to 11.5.

The term “primer,” as used herein, generally refers to a short linearoligonucleotide that hybridizes to a target nucleic acid sequence (e.g.,a DNA template to be amplified) to prime a nucleic acid synthesisreaction. The primer may be an RNA oligonucleotide, a DNAoligonucleotide, or a chimeric sequence. The primer may contain natural,synthetic, or nucleotide analogues (e.g., those that increase T_(m)).Both the upper and lower limits of the length of the primer areempirically determined. The lower limit on primer length is the minimumlength that is required to form a stable duplex upon hybridization withthe target nucleic acid under nucleic acid amplification orpolymerization reaction conditions. Very short primers (usually lessthan 3 nucleotides long) do not form thermodynamically stable duplexeswith target nucleic acid under such hybridization conditions. The upperlimit is often determined by the possibility of having a duplexformation in a region other than the pre-determined nucleic acidsequence in the target nucleic acid. Generally, suitable primer lengthsare in the range of about 3 nucleotides long to about 40 nucleotideslong. The “primers” used in the methods of amplification orpolymerization of a target nucleic acid described herein will be of alength appropriate for a particular set of experimental conditions. Thedetermination of primer length is well within the routine capabilitiesof those of skilled in the art.

The terms “polynucleotide,” “oligonucleotide,” or “nucleic acid,” asused herein, are used herein to refer to biological molecules comprisinga plurality of nucleotides. Exemplary polynucleotides includedeoxyribonucleic acids, ribonucleic acids, and synthetic analoguesthereof, including peptide nucleic acids.

II. Methods of Detection

Described herein are methods for detecting the presence or absence of atleast one polynucleotide analyte in a sample. Detection methods providedherein may use an amplification or polymerization technique (e.g.,polymerase chain reaction (PCR) or PCR process) to incorporatechromophores directly onto the product (e.g., analyte) templates. Anexemplary detection method is shown in FIG. 1, in which a pair ofprimers, each attached to either a fluorophore or a quencher at its 5′end respectively, is used to detect an analyte by amplification orpolymerization process. In some cases, a first signal is measured priorto the start of the amplification or polymerization reaction. During theinitial amplification or polymerization cycle, a duplex DNA separates,allowing primers to bind to specific regions of the individual templatestrands (FIGS. 1A-1C). A polymerase (e.g. Taq polymerase) can be used toextend the primers along the template strand (FIG. 1D). In some cases, asecond signal can be measured after the initial cycle. In some cases, achange in signal is not observed after the initial cycle (FIG. 1E) sincea single chromophore is incorporated into the template. In some cases,upon completion of a second cycle, a change in signal can be observeddue to the incorporation of both chromophores in the synthesizedtemplate (FIG. 1F). In some cases, a second signal can be measured afterthe second cycle. In some cases, the first and second signals are usedto detect the presence or absence of the amplified product (e.g. thepolynucleotide analyte). In some cases, a change is observed between thefirst and second signal. In some cases, the change in signal indicatesthe presence of the amplified product (e.g. the polynucleotide analyte).

In some cases, the change in signal is an increase in signal (e.g., anincreased quenching of fluorescence when a fluorophore and quencher areincorporated into the amplified product). In some cases, the change insignal is a decrease in signal (e.g., a decrease in fluorescenceintensity). In some cases, an increase in signal indicates a presence ofthe product or analyte. In some cases, a decrease in signal indicates apresence of the product or analyte. In some cases, the lack of a changein signal (e.g., no significant change in fluorescence intensity)indicates the absence of the product or analyte. In some cases, adenaturation signal is measured before, during, or after thedenaturation step. In some cases, an annealing signal is measuredbefore, during, or after the annealing step. In some cases, an annealingsignal is measured before, during, or after the extension step. In somecases, the denaturation signal is compared with a reference signal orreference signal range as described elsewhere herein to determine if theamplification or polymerization reaction has proceeded reliably orcorrectly. In some cases, when the denaturation signal falls outside ofthe reference signal range, it can indicate that the reaction hasfailed. In some cases, when the denaturation signal falls within thereference signal range, the denaturation signal can be used to normalizethe annealing signal. In some cases, the denaturation signal from eachcycle can be used for a cycle-by-cycle normalization of the annealingsignals (e.g. chopper stabilization). In some cases, the change inannealing signals is referred to as a relative quantitation of signals.In some cases, the signal is not limited to a signal generated by afluorophore and quencher pair. In some cases, the signal can begenerated by different chromophores.

In some cases, the change in signal can be defined by a percentagechange. In some cases, the change in signal can be about 0.001%, 0.01%,0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%,400%, 500%, 600%, 700%, 800%, 900%, 1000%, or more. In some cases, thechange in signal can be greater than 0.001%, 0.01%, 0.1%, 1%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%,700%, 800%, 900%, or 1000%. In some cases, the change in signal can beless than 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or1000%. In some cases, the change in signal can be about 50%.

Various combinations of oligonucleotides (e.g. primers and/or probes)can be used to detect an analyte. In some cases, a primer is used with aprobe or a plurality of probes. In some cases, a plurality of primers isused with a plurality of probes. In some cases, a plurality of primersis used with a single probe. In some cases, a primer pair is used todetect an analyte. In some cases, a primer pair is used to detectmultiple analytes. In some cases, multiple primer pairs are used todetect an analyte. In some cases, a primer and a probe is used to detectan analyte. In some cases, a primer and a probe is used to detectmultiple analytes. In some cases, a combination of primers and probes isused to detect an analyte. In some cases, a combination of primers andprobes is used to detect multiple analytes. In some cases, a probe isused to detect an analyte. In some cases, a probe is used to detectmultiple analytes. In some cases, multiple probes are used to detect ananalyte.

In some cases, at least one chromophore (e.g. fluorophore, quencher,intercalating dye) is used to detect an analyte. In some cases, achromophore pair (e.g. fluorophore/quencher pairs) is used to detect ananalyte. In some cases, a single chromophore pair is used to detect ananalyte. In some cases, a single chromophore pair is used to detectmultiple analytes. In some cases, multiple chromophore pairs are used todetect an analyte. In some cases, a chromophore pair comprises a firstchromophore and a second chromophore. In some cases, each primer in aprimer pair comprises either a first chromophore (e.g. a fluorophore) ora second chromophore (e.g. a quencher). In some cases, each primer inthe primer pair does not comprise both first and second chromophores. Insome cases, a chromophore pair is not used to detect an analyte. In somecases, a chromophore is not used to detect an analyte. In some cases, achromophore is an intercalating dye. In some cases, an intercalating dyeis not used to detect an analyte. In some cases, the first chromophoreand the second chromophore are the same.

In some cases, a plurality of primers is used in the detection methods.In some cases, a primer is used with an oligonucleotide (e.g. a probe)or a plurality of oligonucleotides (e.g. a plurality of probes). In somecases, a primer is used with a probe or a plurality of probes. In somecases, a plurality of primers is used with a plurality of probes. Insome cases, a plurality of primers is used with a single probe. In somecases, the number of primers used equals the number of probes used inthe detection methods. In some cases, the primer is not attached to theoligonucleotide.

In some cases, a probe is not used in the detection methods. In somecases, a probe with chromophores attached is not used in the detectionmethods. In some cases, a probe (such as a self-quenching probe) with afluorophore and quencher pair attached is not used in the detectionmethods. In some cases, a probe with a fluorophore attached at its 5′end and a quencher attached at its 3′ end is not used in the detectionmethods.

In some cases, the method is used with a second method. In some cases,the second method is an amplification or polymerization method, anelectrophoresis (e.g. gel electrophoresis, capillary electrophoresis), amass spectroscopy method, a chromatography method or an assay (e.g. invitro cell based assay). In some cases, the amplification orpolymerization method is an isothermal reaction method or a polymerasechain reaction method. In some cases, the polymerase chain reactionprocess is a multiplex-PCR, a quantitative PCR (qPCR), an end point PCRor a digital PCR (e.g. droplet digital PCR) process. In some cases, thepolymerase chain reaction is a droplet digital PCR. In some cases, thesecond method is a droplet digital PCR method. In some cases, the secondmethod is an electrophoresis method (e.g. a gel electrophoresis methodfor DNA sequencing). In some cases, the method is used in combinationwith a second and a third method. In some cases, the third method is anamplification or polymerization method, an electrophoresis (e.g. gelelectrophoresis, capillary electrophoresis), a mass spectroscopy method,a chromatography method or an assay (e.g. in vitro cell based assay). Insome cases, the method is used without a second or a third method. Insome cases, the method is used without a second method. In some cases,the method is used without an electrophoresis method. In some cases, themethod is used without a sequencing method.

A. Detection of Analytes from Intensity-Length Relationship

Described herein is a method of detecting the presence of one or moreanalytes in a sample. Methods provided herein involve, e.g., themeasurement of the change in signal intensity when at least twochromophores interact with each other. For example, in the case of afluorophore and quencher interaction, the further the fluorophore isfrom the quencher, the brighter the fluorescence signature of aparticular nucleic acid analyte will typically be. So long that theanalyte lengths are small, the persistence length of the nucleic acidanalyte typically determines the intensity of fluorescence. For example,for a given fluorophore-quencher pair, the intensity of the fluorescencecan be correlated with the persistence length. Further, the intensity offluorescence often indicative of an energy transfer between thefluorophore and the quencher. The efficiency of this energy transfer isdescribed by the following equation:

$\begin{matrix}{E = \frac{1}{1 + \left( {r\text{/}R_{0}} \right)^{6}}} & (1)\end{matrix}$where r is the distance between the fluorophore and the quencher (FIG.2) and R₀ is a constant related to each fluorophore/quencher pair whereit can be calculated from certain parameters of the absorption andemission spectra of each chromophore. (See Biophysical Chemistry, D.Freifelder, ed., W.H. Freeman and Company, San Francisco (1976) at page426-28). Further, R₀ is described by the following equation:

$\begin{matrix}{R_{0}^{6} = \frac{9\;{Q_{0}\left( {\ln\mspace{14mu} 10} \right)}\kappa^{2}J}{128\;\pi^{5}n^{4}N_{A}}} & (2)\end{matrix}$where Q₀ is the fluorescence quantum yield of the donor in the absenceof the acceptor, κ² is the dipole orientation factor, n is therefractive index of the medium, N_(A) is Avogadro's number and J is thespectral overlap integral (FIG. 2).

Therefore, the changes in fluorescence signal typically vary with thelength of the synthesized strand. In some cases, a decrease influorescence signal is inversely proportional with the length of thesynthesized strand. Utilizing this relationship, the presence of ananalyte can be detected based on the signal correlated with its length.For example, a set of fluorescence signal ladders reminiscent ofmolecular weight ladders based on DNA length can be established as acontrol. In a sample, multiple pairs of primers attached with the samefluorophore/quencher pair are amplified. Upon completion of theamplification or polymerization process, the observed signals can becorrelated with the controls, thereby detecting the presence or absenceof a particular analyte. In some cases, the presence or absence of aparticular analyte can be monitored throughout the amplification orpolymerization process, by taking measurements during each amplificationor polymerization cycle and comparing with the control ladder. In somecases, the ladder comprises a plurality of signals. In some cases, theplurality of signals generates multiple curves. In some cases, theladder is represented by a plurality of curves. In some cases, theladder comprises multiple sets of endpoint fluorescence. In some cases,the ladder comprises multiple sets of initial and end pointfluorescence. In some cases, each step of the ladder comprises aplurality of signals. In some cases, the plurality of signals generatesa curve. In some cases, each step of the ladder is represented by acurve. In some cases, each step of the ladder comprises an endpointfluorescence measurement. In some cases, each step of the laddercomprises a set of initial and endpoint fluorescence measurements. Insome cases, each step of the ladder comprises an initial and endpointfluorescence. In some cases, the curve represents a signature profile ofan analyte based on its length. In some cases, the endpoint fluorescencerepresents a signature profile of an analyte based on its length. Insome cases, the set of initial and endpoint fluorescence represents asignature profile of an analyte based on its length. In some cases, theinitial and endpoint fluorescence represents a signature profile of ananalyte based on its length. In some cases, each step of the laddergenerates a signature profile of an analyte based on its length. In somecases, the ladder comprises multiple steps or multiple signatureprofiles of analytes. In some cases, the ladder comprises a single stepor a single signature profile of an analyte. In some cases, the laddercomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, or more steps. In some cases, theladder comprises more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 steps. In somecases, the ladder comprises less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 steps. Insome cases, multiple analytes are detected by a singlefluorophore/quencher pair. In some cases, a single analyte is detectedby a single fluorophore/quencher pair. In some cases, the signal is notlimited to a signal generated by a fluorophore and quencher pair. Insome cases, the signal can be generated by different chromophores.

B. Detection of Genetic Variations

Disclosed herein is a method of determining the presence or absence of agenetic variation, e.g., based on the change in signal due, e.g., to adisruption in the electron transport mechanism described herein. Geneticvariations include deletion and insertion of one or more nucleotides,translocations of different nucleotide occurrences (e.g. single pointmutations such as SNPs or a base-pair substitution), or variations inthe number of multiple nucleotide repetitions. For example, to detectthe presence of a single deletion or alteration (e.g. a SNP) in atemplate (e.g. an analyte), a first primer is designed to hybridize to aregion comprising the deletion. A second primer comprises a sequencecomplementary to the region of the analyte about less than 500 bp awayfrom the first primer. Upon amplification or polymerization, a change insignal is observed. However, since a kink is present in the producttemplate, an inefficient electron transport results in a decrease in thechange of signal, e.g., when compared to the change in signal observedfor an analyte without the genetic variation.

In some cases, the genetic variation detected is a different nucleotideoccurrence in the analyte. In some cases, the different nucleotideoccurrence is a single-nucleotide polymorphism (SNP). A SNP is a DNAsequence variation that occurs when a single nucleotide (e.g. A, T, C orG) in the genome is altered. In some cases, this alteration leads toeither a presence of disease or is associated with (or a marker for) thepresence of a disease or diseases. For example, a single nucleotidemutation from GAG to GTG in the β-globin gene that encodes haemoglobinresults in development of sickle-cell anaemia.

In general, each individual has many SNPs that create a unique human DNApattern. In some cases, a SNP is a common SNP or a rare SNP. In somecases, a SNP is a common SNP. In some cases, a common SNP has a minorallele frequency of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%,12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more. In some cases, acommon SNP has a minor allele frequency of greater than 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or20%. In some cases, a common SNP has a minor allele frequency of lessthan 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,16%, 17%, 18%, 19%, or 20%. In some cases, a SNP is a rare SNP. In somecases, a rare SNP has a minor allele frequency of about 1%, 2%, 3%, 4%,5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,20%, or more. In some cases, a rare SNP has a minor allele frequency ofgreater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some cases, a rare SNP has aminor allele frequency of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

In some cases, provided herein is a method to detect the presence of aSNP. In some cases, the method described herein is used to detect thepresence of a common SNP. In some cases, the method described herein isused to detect the presence of a rare SNP. In some cases, the methoddescribed herein is used to detect the presence of a combination ofcommon and rare SNPs.

In some cases, the method described herein is used to detect thepresence of SNP in a sample. In some cases, the method described hereinis used to detect multiple SNPs in a sample. In some cases, the methoddescribed herein is used to detect multiple common SNPs in a sample. Insome cases, the method described herein is used to detect multiple rareSNPs in a sample. In some cases, the method described herein is used todetect a combination of common and rare SNPs in a sample. In some cases,the method described herein is used to detect a single SNP in a sample.In some cases, the method described herein is used to detect a singlecommon SNP in a sample. In some cases, the method described herein isused to detect a single rare SNP in a sample.

In some cases, a plurality of SNPs is detected in a sample using asingle chromophore pair (e.g. fluorophore/quencher pair). In some cases,the plurality of SNPs can be detected based in part on the length ofeach analyte containing a specific SNP. In some cases, a set offluorescence ladder comprising SNP analytes (an analyte containing atleast one SNP) of known length as described herein is generated. In somecases, the set of fluorescence ladder is used as a control. In somecases, the presence or absence of a particular SNP analyte can bedetermined by taking measurements during the amplification orpolymerization reaction and comparing with the control ladder. In somecases, the presence or absence of a particular SNP analyte can bemonitored throughout the amplification or polymerization process, bytaking measurements during each amplification or polymerization cycleand comparing with the control ladder. In some cases, a plurality ofSNPs is detected in a sample using a relative quantification method.

In some cases, the presence of SNPs correlates directly with thedevelopment of a disease. In some cases, the presence of SNPs increasesthe chances of developing a disease. In some cases, the diseasecomprises a genetic disorder, an autoimmune disease, a neurologicaldisease, a cardiovascular disease and cancer.

C. Monitoring an Amplification or Polymerization Reaction

Disclosed herein is a method for detecting a change in signal generatedby a set of chromophores for monitoring a reaction. In some cases, themethod described herein can be used to monitor the progress of a PCRreaction. For example, at cycle 1, a set of fluorescence signals aremeasured, one measurement at the denaturing step and one measurement atthe annealing step. During cycle 2, a second set of fluorescence signalsare measured at the denaturing and annealing steps. A change influorescence between the signals taken at the two annealing stepindicate an occurrence of a PCR reaction, while the signals taken duringthe denaturing steps are used both as a control and a normalizationparameter.

In some cases, signals measured from the annealing steps are used tomonitor the progress of a reaction. In some cases, the change in signalfrom two annealing steps is measured. The change in signal can be, e.g.,an increase in signal or a decrease in signal. In some cases, the changein signal is defined by a percentage change. In some cases, the changein signal can be about 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000% or more. In some cases, the change in signal can be greaterthan 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000%. Insome cases, the change in signal can be less than 0.001%, 0.01%, 0.1%,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, 600%, 700%, 800%, 900% or 1000%. In some cases, signals aremeasured before, during or after the annealing step.

In some cases, signals measured from the extension steps are used tomonitor the progress of a reaction. In some cases, the change in signalfrom two extension steps is measured. The change in signal can be, e.g.,an increase in signal or a decrease in signal. In some cases, the changein signal is defined by a percentage change. In some cases, the changein signal can be about 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%,900%, 1000%, or more. In some cases, the change in signal can be greaterthan 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000%. Insome cases, the change in signal can be less than 0.001%, 0.01%, 0.1%,1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,500%, 600%, 700%, 800%, 900%, or 1000%. In some cases, the signals aremeasured before, during or after the extension step.

In some cases, the signals measured from the denaturing steps are usedto monitor the progress of a reaction. In some cases, the denaturationsignals are compared with a reference signal or reference signal range.As described elsewhere herein, when a denaturation signal is outside ofthe reference signal range, the denaturation signals can indicate thatthe reaction has failed. In some cases, when the denaturation signal iswithin the reference signal range, the denaturation signals can indicatethat the reaction has succeeded. In some cases, the denaturation signalsserve as a control for each amplification or polymerization cycle. Insome cases, the denaturation signal is used for normalization during anamplification or polymerization experiment. As described elsewhereherein, when the denaturation signal is within the reference signalrange, the denaturation signal from each amplification or polymerizationcycle can be used to normalize the annealing signal from that cycle. Insome cases, the denaturation signals are used for cycle-by-cycleself-normalization of a reaction. In some cases, signals are measuredbefore, during or after the denaturation step.

In some cases, multiple reactions are monitored. In some cases, multiplereactions from a single sample are monitored. In some cases, multiplereactions from multiple samples are monitored. In some cases, a singlereaction is monitored. In some cases, a single reaction from a singlesample is monitored. In some cases, the reaction is used to detect thepresence of a genetic variation. In some cases, the reaction fordetecting the presence of a genetic variation is monitored. In somecases, the reaction is used to detect the presence of a SNP. In somecases, the reaction for detecting the presence of a SNP is monitored. Insome cases, multiple oligonucleotides (e.g. primers and/or probes) areused. In some cases, multiple primers are used. In some cases, multipleprobes are used. In some cases, the number of primers equals the numberof probes.

D. Detection of Morphological Change

Disclosed herein is a method for detecting or monitoring a morphologicalchange in an analyte based on changes in signals. In some cases, ananalyte is a protein, a polynucleotide, a lipid, a carbohydrate or anantibody. In some cases, an analyte is a polynucleotide. In some cases,the polynucleotide is a DNA or a RNA. In some cases, DNA and RNA canadopt different conformations such as a hairpin, tetraloop orpseudoknot. For example, to detect the different morphological state ofa DNA containing a hairpin, a fluorophore/quencher pair can be attachedto the respective stem of the hairpin. Since the fluorophore is in closeproximity to the quencher, a signal may not be observed. As thetemperature increases, the DNA hairpin unwinds and a fluorescence signalmay be observed. In some cases, multiple signals are measured as the DNAunwinds. In some cases, only an initial and an end-point signals aremeasured as the DNA unwinds. In some cases, the multiple signals cangenerate a curve. In some cases, the multiple signals are used togenerate a signature profile of a DNA containing a hairpin. In somecases, the signature profile is a curve. In some cases, an initial andan end-point signals are used to generate a signature profile. In somecases, the signature profile obtained from the DNA denaturation study isused as a control to detect the presence of a hairpin in a target DNA.In some cases, the method described herein is used to monitor thestability of a DNA or RNA conformation after introduction of addition,deletion, substitution or base modifications (e.g. unnatural bases)within the DNA or RNA. In some cases, the stability is affected byexternal factors. In some cases, the external factors include pH,organic or inorganic agents (e.g. salt, intercalating dye) or additionalanalytes. In some cases, the additional analyte is a DNA, RNA, proteinor an antibody. In some cases, the method described herein is used tomonitor the stability of a DNA or RNA conformation after introduction ofthe external factors.

In some cases, the method described herein is used to monitor amorphological change of a protein. For example, a protein residing in anative state can be a folded protein, a partially folded protein or adisordered protein. Folding or unfolding occurs due to the presence ofbinding partners, organic or inorganic agents, pH, and temperature. Fora folded protein, an increase in temperature induces the protein toundergo an unfolding state. By attaching proteins to a plurality offluorophores and/or quenchers, a fluorescence signal can be measuredwith each iterative temperature increase and can be compared to thesignals taken at its native state. In some cases, multiple signals aremeasured as the protein unfolds. In some cases, only an initial and anend-point signals are measured as the protein unfolds. In some cases,multiple signals can generate a curve. In some cases, multiple signalsare used to generate a signature profile of the protein. In some cases,the signature profile is a curve. In some cases, an initial and anend-point signals are used to generate a signature profile. In somecases, the signature profile obtained from the protein unfolding studyis used as a control to detect the morphology of proteins containingsimilar folds. In some cases, the method described herein is used tomonitor the stability of a protein. In some cases, unfolding of theprotein can be induced upon addition of an external factor. In somecases, the external factors include pH, organic or inorganic agents(e.g. salt, intercalating dye) or additional analytes. In some cases,the additional analyte is a DNA, RNA, protein, or an antibody.

In some cases, the method described herein is used to monitor themorphology of an analyte-analyte interaction such as a protein-protein,protein-antibody or protein-polynucleotide (e.g. protein-DNA orprotein-RNA) interactions. For example, during a protein-DNAinteraction, a protein can adopt a different conformation upon bindingof the DNA. In some cases, the change in signal associated with bindingcan be used to compare with the protein at its apo or unbound state. Insome cases, multiple signals are measured as the protein-DNA complexforms. In some cases, only an initial and an end-point signals aremeasured as the complex forms. In some cases, the multiple signals cangenerate a curve. In some cases, the multiple signals are used togenerate a signature profile of the protein. In some cases, thesignature profile is a curve. In some cases, an initial and an end-pointsignals are used to generate a signature profile. In some cases, thesignature profile obtained from the protein-DNA study is used as acontrol to detect the formation of protein complex with additional DNAs.In some cases, the methods described herein can monitor the stability ofthe protein complex with addition of another external factor. In somecases, the methods described herein can be used to monitor themorphological change of an analyte with multiple binding partners.

III. Multiplex Detection

Disclosed herein are examples of determining the presence of a pluralityof analytes using a plurality of chromophores to indicate the presenceor absence of these analytes. For example, a multiplex detection methodcan combines the use of color, signal, and/or mathematical strategies tocircumvent degeneracy and ensure an infinite number of unique codes thatcan be unambiguously decoded in any combination of occurrences. Forexample, in detecting a sample containing four analytes, each analytecan be assigned a fluorophore (blue, green, yellow, or red) and aquencher attached to analyte-specific oligonucleotides (e.g., a forwardPCR primer and a reverse PCR primer). Upon amplification orpolymerization, the presence or absence of an analyte is determinedbased on the presence or absence of a signal in that particular color.

In some cases, multiple color codes are generated using a plurality ofchromophores. In some cases, multiple color codes are generated using aplurality of fluorophores and quenchers. In some cases, multiple colorcodes are generated using combinations of fluorophore/quencher pairs. Insome cases, multiple chromophores are assigned to multiple analytes. Insome cases, a single chromophore is assigned to multiple analytes. Insome cases, a single chromophore is assigned to one analyte.

In some cases, one color code or chromophore combination is assigned toone analyte. In some cases, one color code or chromophore combination isassigned to multiple analytes (e.g., to discriminate multiple analytesof varying lengths in a single detection reaction). In some cases, onecolor code or chromophore combination is assigned to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10,000, or 100,000analytes. In some cases, multiple color codes or chromophorecombinations are assigned to one or more analytes. In some cases, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10,000, or100,000 color codes or chromophore combinations are assigned to one ormore analytes (e.g. 1, 5, 10, 100, 500, 10,000 analytes). In some cases,at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000,5000, 10,000, or 100,000 color codes or chromophore combinations areassigned to one or more analytes (e.g. 1, 5, 10, 100, 500, 10,000analytes).

In some cases, one color is assigned as a control. In some cases, thecontrol is a positive control or a negative control.

A. Multiplex Detection for Genetic Variation

In some cases, the methods disclosed herein can be used to detect thepresence of multiple genetic variations (e.g., SNPs). In some cases, ananalyte contains a plurality of genetic variations. In some cases, ananalyte contains one genetic variation. In some cases, one color isassigned to one genetic variation. In some cases, a sample contains aplurality of genetic variations, wherein a color code or chromophorecombination is assigned to each genetic variation. In some cases, asample contains one genetic variation.

B. Multiplex Detection for SNP

In some cases, disclosed herein are methods of detecting the presence orabsence of a SNP in an analyte. In some cases, an analyte contains aplurality of SNPs. In some cases, an analyte contains 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, 500, or more SNPs. In some cases, an analyte contains at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, or 500 SNPs. In some cases, an analyte contains no morethan 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, or 500 SNPs. In some cases, an analyte contains1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, 500, or more common SNPs. In some cases, an analytecontains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 250, 300, 350, 400, 450, or 500 common SNPs. In some cases, ananalyte contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, or 500 common SNPs. Insome cases, an analyte contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, or more rareSNPs. In some cases, an analyte contains at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400,450, or 500 rare SNPs. In some cases, an analyte contains no more than1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250,300, 350, 400, 450, or 500 rare SNPs.

In some cases, a sample contains a plurality of analytes. In some cases,multiple SNPs are detected from a plurality of analytes in the sample.In some cases, multiple common SNPs are detected from a plurality ofanalytes in the sample. In some cases, multiple rare SNPs are detectedfrom a plurality of analytes in the sample. In some cases, multiple SNPsare detected from an analyte in the sample. In some cases, multiplecommon SNPs are detected from an analyte in the sample. In some cases,multiple rare SNPs are detected from an analyte in the sample. In somecases, one SNP is detected from an analyte in the sample. In some cases,one common SNP is detected from an analyte in the sample. In some cases,one rare SNP is detected from an analyte in the sample. In some cases,one SNP is detected in the sample. In some cases, one common SNP isdetected in the sample. In some cases, one rare SNP is detected in thesample.

In some cases, a sample contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more SNPs. In somecases, a sample contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more SNPs. In somecases, a sample contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more SNPs. In somecases, a sample contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 5000, 10,000, 50,000, 100,000, or more common SNPs. In some cases,a sample contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 5000, 10,000, 50,000, 100,000, or more common SNPs. In somecases, a sample contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more common SNPs. Insome cases, a sample contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 5000, 10,000, 50,000, 100,000, or more rare SNPs. In somecases, a sample contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more rare SNPs. Insome cases, a sample contains no more than 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more rareSNPs.

In some cases, the methods described herein comprise detecting 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000,100,000, or more SNPs. In some cases, the methods described hereincomprise detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800,900, 1000, 5000, 10,000, 50,000, 100,000, or more SNPs. In some cases,the methods described herein comprise detecting no more than 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000, 100,000,or more SNPs. In some cases, the methods described herein comprisedetecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000,10,000, 50,000, 100,000, or more common SNPs. In some cases, the methodsdescribed herein comprise detecting at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more commonSNPs. In some cases, the methods described herein comprise detecting nomore than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000,10,000, 50,000, 100,000, or more common SNPs. In some cases, the methodsdescribed herein comprise detecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more rare SNPs. Insome cases, the methods described herein comprise detecting at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300,350, 400, 450, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 50,000,100,000, or more rare SNPs. In some cases, the methods described hereincomprise detecting no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 5000, 10,000, 50,000, 100,000, or more rare SNPs.

In some cases, the method of detection utilizes an amplification orpolymerization method. In some cases, an amplification or polymerizationmethod (or process) comprises a polymerase chain reaction (PCR) methodand an isothermal reaction method. In some cases, a PCR reaction processcomprises a multiplex PCR, a real-time PCR, a quantitative PCR and adigital PCR (e.g. droplet digital PCR) process. In some cases, themethod of detection utilizes a quantitative PCR method. In some cases,the quantitative PCR method is used in combination with a second method.In some cases, the second method is a digital PCR method. In some cases,the second method is a droplet digital PCR method.

In some cases, a signature profile is used to detect the presence of aSNP. In some cases, a signature profile is used to pinpoint thenucleotide mutation. In some cases, a signature profile is unique foreach nucleotide mismatch, UU, UT, UG, UC, UA, AA, TT, GG, CC, AG, AC,TC, TC, and distinct from the wild-type. In some cases, a signatureprofile of a nucleotide mismatch is compared to that of a wild-type. Insome cases, the signature profile of a SNP is compared to that of awild-type. In some cases, a fluorescence signal of a SNP is compared toa fluorescence signal of a wild-type. In some cases, a change influorescence signal is detected between the signals of a SNP and awild-type. In some cases, the change in signal can be calculated as apercentage. In some cases, the percentage of signal change is 0.01, 0.1,0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100%. In some cases, the percentage of signalchange is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In some cases,a change in signal is detected between the fluorescence signals of an AGmismatch and a wild-type. In some cases, the change in signal iscalculated as a percentage. In some cases, the percentage of signalchange is 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In some cases, thepercentage of signal change is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or100%. In some cases, a change in signal is detected between thefluorescence signals of an AC mismatch and a wild-type. In some cases,the change in signal is calculated as a percentage. In some cases, thepercentage of signal change is 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%.In some cases, the percentage of signal change is about 0.01, 0.1, 0.2,0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100%. In some cases, a change in signal is detectedbetween the fluorescence signals of a TG mismatch and a wild-type. Insome cases, the change in signal is calculated as a percentage. In somecases, the percentage of signal change is 0.01, 0.1, 0.2, 0.5, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90,95, or 100%. In some cases, the percentage of signal change is about0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, or 100%. In some cases, a change insignal is detected between the fluorescence signals of a TC mismatch anda wild-type. In some cases, the change in signal is calculated as apercentage. In some cases, the percentage of signal change is 0.01, 0.1,0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, or 100%. In some cases, the percentage of signalchange is about 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%.

In some cases, a pair of primers is utilized to detect a SNP. In somecases, the first primer comprises a sequence encoding the SNP. In somecases, the first primer hybridizes to a region of the analyte encodingthe SNP. In some cases, the second primer comprises a sequence notencoding the SNP. In some cases, the second primer comprises a sequencecomplementary to a region of the analyte not encoding the SNP. In somecases, the first primer encodes a region on the analyte greater than 500base pairs apart from a region of the analyte encoded by the secondprimer. In some cases, the first primer encodes a region on the analyteless than 500 base pairs apart from a region encoded by the secondprimer. In some cases, the first primer encodes a region on the analyteless than 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380,370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240,230, 220, 210, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150,145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95,94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77,76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59,58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41,40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,22, 21, 20 base pairs apart from a region encoded by the second primer.In some cases, the first primer encodes a region on the analyte no morethan 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370,360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230,220, 210, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145,140, 135, 130, 125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95, 94, 93,92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75,74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57,56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39,38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21,20 base pairs apart from a region encoded by the second primer. In somecases, the first primer encodes a region on the analyte about 490, 480,470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340,330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200,195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130,125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90,89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72,71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54,53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 basepairs apart from a region encoded by the second primer.

IV. Analytes

An analyte may be any suitable analyte that can be analyzed using themethods and compositions of the present disclosure, where the analyte iscapable of interacting with a reagent (e.g., an oligonucleotide such asa primer or probe attached to a chromophore) in order to generate asignal that can be measured. An analyte may be naturally-occurring orsynthetic. An analyte may be present in a sample obtained using anymethods known in the art. In some cases, a sample may be processedbefore analyzing it for an analyte. The methods and compositionspresented in this disclosure may be used in solution phase assays,without the need for particles (such as beads) or a solid support.

In some cases, an analyte may be a polynucleotide, such as DNA, RNA,peptide nucleic acids, and any hybrid thereof, where the polynucleotidecontains any combination of deoxyribo- and/or ribo-nucleotides.Polynucleotides may be single stranded or double stranded, or containportions of both double stranded or single stranded sequence.Polynucleotides may contain any combination of nucleotides or bases,including, for example, uracil, adenine, thymine, cytosine, guanine,inosine, xanthine, hypoxanthine, isocytosine, isoguanine, and anynucleotide derivative thereof. As used herein, the term “nucleotide” mayinclude nucleotides and nucleosides, as well as nucleoside andnucleotide analogs, and modified nucleotides, including both syntheticand naturally occurring species. Polynucleotides may be any suitablepolynucleotide for which one or more reagents as described herein may beproduced, including but not limited to cDNA, mitochondrial DNA (mtDNA),messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), nuclearRNA (nRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA),small nucleolar RNA (snoRNA), small Cajal body-specific RNA (scaRNA),microRNA (miRNA), double stranded (dsRNA), ribozyme, riboswitch, orviral RNA. Polynucleotides may be contained within any suitable vector,such as a plasmid, cosmid, fragment, chromosome, or genome. In somecases, the analyte is referred to as a polynucleotide analyte. In somecases, the analyte is referred to as a nucleic acid analyte. In somecases, the analyte is referred to as a nucleic acid target.

Genomic DNA may be obtained from naturally occurring or geneticallymodified organisms or from artificially or synthetically createdgenomes. Analytes comprising genomic DNA may be obtained from any sourceand using any methods known in the art. For example, genomic DNA may beisolated with or without amplification or polymerization. Amplificationor polymerization may include PCR amplification, multiple displacementamplification (MDA), rolling circle amplification and otheramplification or polymerization methods. Genomic DNA may also beobtained by cloning or recombinant methods, such as those involvingplasmids and artificial chromosomes or other conventional methods (seeSambrook and Russell, Molecular Cloning: A Laboratory Manual., citedsupra.) Polynucleotides may be isolated using other methods known in theart, for example as disclosed in Genome Analysis: A Laboratory ManualSeries (Vols. I-IV) or Molecular Cloning: A Laboratory Manual. If theisolated polynucleotide is an mRNA, it may be reverse transcribed intocDNA using conventional techniques, as described in Sambrook andRussell, Molecular Cloning: A Laboratory Manual., cited supra.

An analyte may be a protein, polypeptide, lipid, carbohydrate, sugar,small molecule, or any other suitable molecule that can be detected withthe methods and compositions provided herein. An analyte may be anenzyme or other protein. An analyte may be a drug or metabolite (e.g.anti-cancer drug, chemotherapeutic drug, anti-viral drug, antibioticdrug, or biologic). An analyte may be any molecule, such as a co-factor,receptor, receptor ligand, hormone, cytokine, blood factor, antigen,steroid, or antibody.

An analyte may be any molecule from any pathogen, such as a virus,bacteria, parasite, fungus, archaea or prion (e.g., PrP^(Sc)). Exemplaryviruses include those from the families Adenoviridae, Flaviviridae,Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae,Paramyxoviridae, Picornaviridae, Polyomavirus, Retroviridae,Rhabdoviridae, and Togaviridae. Specific examples of viruses includeadenovirus, astrovirus, bocavirus, BK virus, coxsackievirus,cytomegalovirus, dengue virus, Ebola virus, enterovirus, Epstein-Barrvirus, feline leukemia virus, hepatitis virus, hepatitis A virus,hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis Evirus, herpes simplex virus (HSV), HSV type 1, HSV type 2, humanimmunodeficiency virus (HIV), HIV type 1, HIV type 2, human papillomavirus (HPV), HPV type 1, HPV type 2, HPV type 3, HPV type 4, HPV type 6,HPV type 10, HPV type 11, HPV type 16, HPV type 18, HPV type 26, HPVtype 27, HPV type 28, HPV type 29, HPV type 30, HPV type 31, HPV type33, HPV type 34, HPV type 35, HPV type 39, HPV type 40, HPV type 41, HPVtype 42, HPV type 43, HPV type 44, HPV type 45, HPV type 49, HPV type51, HPV type 52, HPV type 54, HPV type 55, HPV type 56, HPV type 57, HPVtype 58, HPV type 59, HPV type 68, HPV type 69, influenza type A virus,influenza type B virus, JC virus, Marburg virus, measles virus,metapneumovirus, mumps virus, Norwalk virus, parovirus, polio virus,rabies virus, respiratory syncytial virus including type A and type B,retrovirus, rhinovirus, rotavirus, Rubella virus, smallpox virus,vaccinia virus, West Nile virus, yellow fever virus, and humanparainfluenza virus type 3.

Exemplary bacteria include those from the genuses Bordetella, Borrelia,Brucella, Campylobacter, Chlamydia, Clostridium, Corynebacterium,Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter,Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus,Streptococcus, Treponema, Vibrio, and Yersinia. Specific examples ofbacteria include Bordetella par apertussis, Bordetella pertussis,Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucellamelitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae,Chlamydia psittaci, Chlamydia trachomatix, Clostridium botulinum,Clostridium difficile, Clostridium perfringens, Clostridium tetani,Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcusfaecium, Escherichia coli, Francisella tularensis, Haemophilusinfluenzae, Helicobacter pylori, Legionella pneumophila, Leptospirainterrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacteriumtuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseriagonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsiarickettsii, Salmonella choleraesuis, Salmonella dublin, Salmonellaenteritidis, Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersiniapestis, and Yersinia enterocolitica.

Exemplary parasites include those from the genuses Acanthamoeba,Babesia, Balamuthia, Balantidium, Blasocystis, Cryptosporidium,Dientamoeba, Entamoeba, Giardia, Isospora, Leishmania, Naegleria,Pediculus, Plasmodium, Rhinosporidium, Sarcocystis, Schistosoma,Toxoplasma, Trichomonas, and Trypanosoma. Specific examples of parasitesinclude Babesia divergens, Babesia bigemina, Babesia equi, Babesiamicrofti, Babesia duncani, Balamuthia mandrillaris, Balantidium coli,Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isosporabelli, Naegleria fowleri, Pediculus humanus, Plasmodium falciparum,Plasmodium knowlesi, Plasmodium malariae, Plasmodium ovale, Plasmodiumvivax, Rhinosporidium seeberi, Sarcocystis bovihominis, Sarcocystissuihominis, Schistosoma mansoni, Toxoplasma gondii, Trichomonasvaginalis, Trypanosoma brucei, and Trypansoma cruzi.

Exemplary fungi include those from the genuses Apophysomyces,Aspergillus, Blastomyces, Candida, Cladosporium, Coddidioides,Cryptococcos, Exserohilum, Fusarium, Histoplasma, Pichia, Pneumocystis,Saccharomyces, Sporothrix, Stachybotrys, and Trichophyton. Specificexamples of fungi include Aspergillus fumigatus, Aspergillus flavus,Aspergillus clavatus, Blastomyces dermatitidis, Candida albicans,Coccidioides immitis, Crytptococcus neoformans, Exserohilum rostratum,Fusarium verticillioides, Histoplasma capsulatum, Pneumocystisjirovecii, Sporothrix schenckii, Stachybotrys chartarum, andTrichophyton mentagrophytes.

Exemplary archaea include those from the genuses Acidilobus,Acidococcus, Aeropyrum, Archaeoglobus, Caldisphaera, Caldococcus,Cenarchaeum, Desulfurococcus, Geogemma, Geoglubus, Haladaptatus,Halomicrobium, Hyperthermus, Ignicoccus, Ignisphaera, Methanobacterium,Natronococcus, Nitrosopumilus, Picrophilus, Pyrodictium, Pyrolobus,Staphylothermus, Stetteria, Sulfophobococcus, Thermodiscus,Thermosphaera and Thermoplasma. Specific examples of archea include A.aceticus, A. camini, A. fulgidus, A. infectus, A. lithotrophicus, A.pernix, A. profundus, A. veneficus, A. saccharovorans, A.sulfurreducens, C. dracosis, C. lagunensis, C. noboribetus, C.symbiosum, D. amylolyticus, D. fermentans, D. mobilis, D. mucosus, G.barossii, G. indica, G. pacifica, H. butylicus, N. maritimus, G.ahangari, H. paucihalophilus, H. mukohataei, H. katesii, H. zhouii, I.aggregans, I. islandicus, I. pacificus, I. hospitalis, M. aarhusense, M.alcaliphilum, M. beijingense, M. bryantii, M. congolense, M. curvum, M.espanolae, M. formicicum, M. ivanovii, M. oryzae, M. palustre, M.subterraneum, M. thermaggregans, M. uliginosum, N. amylolyticus, N.jeotgali, N. occultus, P. abyssi, P. brockii, P. occultum, P. fumarii,P. oshimae, P. torridus, S. hellenicus, S. marinus, S. hydrogenophila,S. zilligii, T. maritimus, T. aggregans, T. acidophilum, T. sp. P61, T.sp. S01, T. sp. S02, T. sp. XT101, T. sp. XT102, T. sp. XT103, T. sp.XT107, and T. volcanium.

In some cases, an analyte may be any molecule derived from a mammal. Insome cases, the mammal is a human, a non-human primate, mouse, rat,rabbit, goat, dog, cat, or cow. In some embodiments, the mammal is ahuman. In some cases, a human is a patient.

In some cases, an analyte may be any molecule derived from a plant. Insome cases, a plant is any of various photosynthetic, eukaryotic,multicellular organisms of the kingdom Plantae characteristicallyproducing embryos, containing chloroplasts, having cellulose cell walls,and lacking the power of locomotion.

In some cases, the methods provided in this disclosure may be used todetect any one of the analytes described above, or elsewhere in thespecification. In some cases the methods provided in this disclosure maybe used to detect panels of the analytes described above, or elsewherein the specification. For example, a panel may comprise an analyteselected from the group consisting of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 500, 1000, 5000, 10,000, 100,000,or more analytes described above or elsewhere in the specification.

An analyte may be obtained from any suitable location, including fromorganisms, whole cells, cell preparations and cell-free compositionsfrom any organism, tissue, cell, or environment. Analytes may beobtained from environmental samples, forensic samples, biopsies,aspirates, formalin fixed embedded tissues, air, agricultural samples,soil samples, petroleum samples, water samples, or dust samples. In someinstances, an analyte may be obtained from bodily fluids which mayinclude blood, urine, feces, serum, lymph, saliva, mucosal secretions,perspiration, central nervous system fluid, vaginal fluid, or semen.Analytes may also be obtained from manufactured products, such ascosmetics, foods, personal care products, and the like. Analytes may bethe products of experimental manipulation including, recombinantcloning, polynucleotide amplification or polymerization, polymerasechain reaction (PCR) amplification or polymerization, isothermalamplification or polymerization, purification methods (such aspurification of genomic DNA or RNA), and synthesis reactions.

More than one type of analyte may be detected in each multiplexed assay.For example, a polynucleotide, a protein, a polypeptide, a lipid, acarbohydrate, a sugar, a small molecule, or any other suitable moleculemay be detected simultaneously in the same multiplexed assay with theuse of suitable reagents. Any combination of analytes may be detected atthe same time.

Detection of an analyte may be useful for any suitable application,including research, clinical, diagnostic, prognostic, forensic, andmonitoring applications. Exemplary applications include detection ofhereditary diseases, identification of genetic fingerprints, diagnosisof infectious diseases, cloning of genes, paternity testing, criminalidentification, phylogeny, anti-bioterrorism, environmentalsurveillance, and DNA computing. For example, an analyte may beindicative of a disease or condition. An analyte may be used to make atreatment decision, or to assess the state of a disease. The presence ofan analyte may indicate an infection with a particular pathogen, or anyother disease, such as cancer, autoimmune disease, cardiorespiratorydisease, liver disease, digestive disease, and so on. The methodsprovided herein may thus be used to make a diagnosis and to make aclinical decision based on that diagnosis. For example, a result thatindicates the presence of a bacterial polynucleotide in a sample takenfrom a subject may lead to the treatment of the subject with anantibiotic.

In some cases, the methods and compositions of the present disclosuremay be used to detect at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700,800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000,100,000, or more analytes. In some cases the methods and compositions ofthe present disclosure may be used to detect about 1-10,000, 1-1000,1-100, 1-50, 1-40, 1-30, 1-20, 1-10, or 1-5 analytes.

In some cases, this disclosure provides assays that are capable ofunambiguously detecting the presence or absence of each of 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350,400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10,000, or 100,000 analytes, in any combination ofpresence or absence, in a single sample volume. In some cases, thisdisclosure provides assays that are capable of unambiguously detectingthe presence or absence of each of at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500,600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10,000, or 100,000 analytes, in any combination of presence orabsence, in a single sample volume. In some cases, this disclosureprovides assays that are capable of unambiguously detecting the presenceor absence of less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900,1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, or 100,000analytes, in any combination of presence or absence, in a single samplevolume.

A. Distance

In one aspect, the methods provided herein may be used to detectpolynucleotide analytes containing about 1-1000 base pairs (bp). In somecases, the methods provided herein may be used to detect polynucleotideanalytes containing 1-500 bp, 10-450 bp, 15-400 bp, 20-350 bp, 25-300bp, 30-250 bp, 35-200 bp, or 40-190 bp. In some cases, the methods maybe used to detect a polynucleotide analyte containing 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, or more base pairs. In somecases, the methods may be used to detect polynucleotide analytecontaining at least 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104,105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118,119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, or 190 base pairs. In some cases, the methods may be used to detectpolynucleotide analyte containing no more than 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141,142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183,184, 185, 186, 187, 188, 189, or 190 base pairs.

B. Sensitivity

In some cases, the methods disclosed herein may be used to detectpolynucleotide analyte at concentrations of about 100 uM to about 1 fM.In some cases, the methods provided herein may be used to detect apolynucleotide analyte at concentrations of about 10 uM-20 fM, 1 uM-40fM, 500 nM-60 fM, 100 nM-70 fM, 50 nM-80 fM, 30 nM-90 fM, 10 nM-100 fM.In some cases, the methods may be used to detect a polynucleotideanalyte at a concentration of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM,3 nM, 2 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200pM, 180 pM, 160 pM, 140 pM, 120 pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM,75 pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25pM, 20 pM, 18 pM, 16 pM, 14 pM, 12 pM, 10 pM, 8 pM, 6 pM, 4 pM, 2 pM, 1pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100fM, 50 fM, 10 fM, 1 fM, 100 aM, 10 aM, or 1 aM. In some cases, themethods may be used to detect a polynucleotide analyte at aconcentration of at least 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3nM, 2 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM,600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200 pM,180 pM, 160 pM, 140 pM, 120 pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM, 75pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25pM, 20 pM, 18 pM, 16 pM, 14 pM, 12 pM, 10 pM, 8 pM, 6 pM, 4 pM, 2 pM, 1pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100fM, 50 fM, 10 fM, 1 fM, 100 aM, 10 aM, or 1 aM. In some cases, themethods may be used to detect a polynucleotide analyte at aconcentration of no more than 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM,3 nM, 2 nM, 1 nM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM, 200pM, 180 pM, 160 pM, 140 pM, 120 pM, 100 pM, 95 pM, 90 pM, 85 pM, 80 pM,75 pM, 70 pM, 65 pM, 60 pM, 55 pM, 50 pM, 45 pM, 40 pM, 35 pM, 30 pM, 25pM, 20 pM, 18 pM, 16 pM, 14 pM, 12 pM, 10 pM, 8 pM, 6 pM, 4 pM, 2 pM, 1pM, 900 fM, 800 fM, 700 fM, 600 fM, 500 fM, 400 fM, 300 fM, 200 fM, 100fM, 50 fM, 10 fM, 1 fM, 100 aM, 10 aM, or 1 aM.

C. Specificity

In some methods provided herein, a primer pair may be specific for oneor a plurality of analytes. In some cases, a primer pair is specific to1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes. In somecases, a primer pair is specific to at least 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 analytes. In some cases, a primer pair is specificto less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 analytes. Insome cases, a primer pair is specific to one analyte. In some cases, aprimer pair is universal to all analytes.

V. Probes and Primers

Some of the methods provided in this disclosure utilize a reagent (e.g.an oligonucleotide such as a primer or a probe that is attached to achromophore) that can generate a signal in the presence of an analyte.Any suitable reagent may be used with the present disclosure. Generally,a reagent will have an analyte-specific component and a component thatgenerates a signal in the presence of the analyte. In some cases, thesereagents are referred to as probes and primers. In some cases, theprobes are hybridization probes. In some cases, the hybridization probesare oligonucleotide probes attached to chromophores. In some cases, theprobes are antibodies that detect an analyte, with a fluorescent labelthat emits or is quenched upon binding of the antibody to an analyte. Insome cases, the reagent is a primer. In some cases, the primer isattached to a chromophore. In some cases, the primer is attached to afluorophore. In some cases, the primer is attached to a quencher.

The methods of the present disclosure may use one or more reagents(e.g., an oligonucleotide such as a primer or a probe that is attachedto a chromophore) to detect the presence or absence of each analyte. Forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more reagentsmay be used to detect the presence or absence of each analyte. In somecases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 reagentsmay be used to detect the presence or absence of each analyte. In somecases, fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 reagentsmay be used to detect the presence or absence of each analyte.

In some cases, a sample is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or more reagents to detect the presence or absence ofall analytes. In some cases, a sample is contacted with at least 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more reagents to detect thepresence or absence of all analytes. In some cases, a sample iscontacted with fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 reagents to detect the presence or absence of all analytes.

As described above, primers attached to a fluorophore or a quencher maybe used to detect the presence of an analyte in a polynucleotideamplification or polymerization assay. The quencher can quench thefluorescence emitted by the fluorophore upon excitation by a lightsource when the quencher and fluorophore are in close proximity. Thesequence of the primer can be designed to be complementary to or maycontain nucleotide mutations to a polynucleotide sequence present in ananalyte, and the primer is capable of hybridizing to the analyte. Thesequence of the primer can also be designed to contain one or morenucleotide variations in a polynucleotide sequence of an analyte, andthe primer is capable of hybridizing to the analyte. A fluorophore canbe attached to the 5′ end of one of a primer pair. A quencher can beattached to the 5′ end of the second primer of the primer pair.Hybridization of the primers may be performed in a nucleic acidamplification or polymerization reaction comprising primers (e.g., apolymerase chain reaction). Upon extension of the primers by a DNApolymerase, the fluorophore and quenchers are incorporated in theamplicon or amplification (or polymerization) product (e.g., ananalyte). The incorporation of the quencher and the fluorophore in thenewly generated amplicon can lead to signal generation (e.g., quenchingof fluorescence intensity from the fluorophore). With each iterativeamplification or polymerization reaction, the fluorescence intensity isreduced by a factor of about 2. The amount of fluorescence detected canbe used to directly determine the amount of analyte present. If noanalyte is present, little or no quenching will be observed.

In some cases, a sample to be analyzed is combined with 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, or more pairs of primers (e.g. a forwardprimer and a reverse primer). In some cases a sample to be analyzed iscontacted with at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,or more pairs of primers. In some cases a sample to be analyzed iscontacted with fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 pairs of primers. In some cases, the number of pairs of primers is2-10, 3-15, 4-20, 3-10, 4-10, 5-10, 6-8, or 6-10. In some cases, asample to be analyzed is contacted with 1 pair of primers.

In some cases, a sample may contain one or more analytes. In some cases,one primer pair may be used to detect the presence or absence of eachanalyte. In some cases, a sample is contacted with 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10,000, or moredifferent pairs of primers with each primer pair detecting a singleanalyte. In some cases, a sample is contacted with at least 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10,000, ormore different pairs of primers with each primer pair detecting a singleanalyte. In some cases, a sample is contacted with fewer than 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, 10,000, ormore different pairs of primers with each primer pair detecting a singleanalyte. In some cases, the number of pairs of primers is 2-10, 3-15,4-20, 3-10, 4-10, 5-10, 6-8, or 6-10.

In some cases, primers may be specific for a particular analyte andcapable of amplifying a region complementary to a probe. In some cases,the number of primers used is equivalent to the number of probes. Inother cases, the number of probes used may exceed the number of primerused. In some cases, the number of primers and probes is defined by aratio. In some cases, the ratio of primer to probe is 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000. In some cases, the ratio of probe to primeris 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000.

As disclosed elsewhere herein, primers may have one or a plurality offluorophores or quenchers per primer. For example, in some cases aprimer may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ormore fluorophores. A primer may comprise at least 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, or 50 fluorophores. A primer may comprise fewerthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 fluorophores.

A primer may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ormore quenchers. A primer may comprise at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, or 50 quenchers. A primer may comprise fewer than 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quenchers.

Attachment of fluorophores and quenchers to a probe or a primer may beperformed in the same reaction or in serial reactions. A series ofreactions may be performed to attach probes or primers to at least onefluorophore and the reaction products may be mixed to generate a mixtureof probes or primers with different fluorophores.

Although many aspects of the present disclosure are exemplified usingnucleic acid-based probes and primers, one of ordinary skill in the artwill readily recognize that other forms of probes and primers would workequally well with the examples described in this disclosure. Forexample, a binding molecule specific to an analyte could be used as aprobe. Non-limiting exemplary binding molecules include an antibodyrecognizing an analyte, and generating a signal in the presence of ananalyte.

The fluorescent labels of the present disclosure may be attached to aprobe or primer at any location. In some cases, a single chromophore isattached to the primer at the 5′ end. In some examples, multiplechromophores are attached to the primer with at least one chromophoreattached at the 5′ end. Methods of chromophore labeling are well definedin the art. See, e.g. Pesce et al, editors, Fluorescence Spectroscopy,Marcel Dekker, New York, (1971); White et al, Fluorescence Analysis: APractical Approach, Marcel Dekker, New York, (1970); and the like.Further, there is extensive guidance in the literature for derivatizingreporter and quencher molecules for covalent attachment via commonreactive groups that can be added to an oligonucleotide. See, e.g. U.S.Pat. Nos. 3,996,345; and 4,351,760. In examples that utilize chromophorelabels as described herein, any suitable labeling techniques may beused.

VI. Chromophores

Chromophores are molecules capable of selective light absorptionresulting in the coloration of these molecule containing compounds. Thecolor arises when a molecule at an excited state releases energy in theform of light with a defined spectrum. Exemplary chromophores include,but are not limited to, a fluorochrome, a non-fluorochrome chromophore,a quencher (e.g. fluorescence quencher and a dark quencher), anabsorption chromophore, a fluorophore, any organic or inorganic dye,metal chelate, or any fluorescent enzyme substrate. In some cases, thechromophore is a fluorochrome. In some cases, the fluorochrome is afluorophore. In some cases, the chromophore is a quencher. In somecases, the chromophore is a dark quencher.

Several chromophores are described in the art, e.g. Berlman, Handbook ofFluorescence Spectra of Aromatic Molecules, 2nd Edition, Academic Press,New York, (1971). In examples that utilize fluorescent labels asdescribed herein, any suitable fluorescent label may be used.

Exemplary fluorophores suitable for use with the present disclosureincludes rhodamine, rhodol, fluorescein, thiofluorescein,aminofluorescein, carboxyfluorescein, chlorofluorescein,methylfluorescein, sulfofluorescein, aminorhodol, carboxyrhodol,chlororhodol, methylrhodol, sulforhodol; aminorhodamine,carboxyrhodamine, chlororhodamine, methylrhodamine, sulforhodamine, andthiorhodamine; cyanine, indocarbocyanine, oxacarbocyanine,thiacarbocyanine, merocyanine, cyanine 2, cyanine 3, cyanine 3.5,cyanine 5, cyanine 5.5, cyanine 7, oxadiazole derivatives,pyridyloxazole, nitrobenzoxadiazole, benzoxadiazole, pyren derivatives,cascade blue, oxazine derivatives, Nile red, Nile blue, cresyl violet,oxazine 170, acridine derivatives, proflavin, acridine orange, acridineyellow, arylmethine derivatives, auramine, crystal violet, malachitegreen, tetrapyrrole derivatives, porphin, phtalocyanine and bilirubin;1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalene sulfonate,2-p-touidinyl-6-naphthalene sulfonate, 3-phenyl-7-isocyanatocoumarin,N-(p-(2-benzoxazolyl)phenyl)maleimide, stilbenes, pyrenes, 6-FAM(Fluorescein), 6-FAM (NHS Ester), Fluorescein dT, HEX, JOE (NHS Ester),MAX, TET, ROX, TAMRA, TARMA™ (NHS Ester), TEX 615, ATTO™ 488, ATTO™ 532,ATTO™ 550, ATTO™ 565, ATTO™ Rho101, ATTO™ 590, ATTO™ 633, ATTO™ 647N,TYE™ 563, TYE™ 665, TYE™ 705 and the like.

Exemplary quenchers suitable for use with the present disclosureincludes black hole quenchers, such as BHQ-0, BHQ-1, BHQ-2, BHQ-3; ATTOquenchers, such as ATTO 540Q, ATTO580Q, and ATTO612Q; Qxl quenchers;Iowa Black FG, Iowa Black RG, Iowa Black FQ and Iowa Black RQ; IRDyeQC-1; 1.4 nm Nanogold; and the like.

The fluorophores that may be used with the disclosure are not limited toany of the fluorophores described herein. For example, fluorophores withimproved properties are continually developed, and these fluorophorescould readily be used with the methods provided in this disclosure. Suchimproved fluorophores include quantum dots, which may emit energy atdifferent wavelengths after being excited at a single wavelength.

A. Chromophore Combinations

In some cases, a plurality of chromophores is attached to anoligonucleotide (e.g. a probe or a primer). In some cases, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, or more chromophores are attached toan oligonucleotide. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or more chromophores are attached to an oligonucleotide.In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50chromophores are attached to an oligonucleotide. In some cases, onechromophore is attached to an oligonucleotide.

In some cases, the oligonucleotide comprises a probe. In some cases, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more chromophores areattached to a probe. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, or more chromophores are attached to a probe. In somecases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50chromophores are attached to a probe. In some cases, one chromophore isattached to a probe.

In some cases, the oligonucleotide is a primer. In some cases, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more chromophores areattached to a primer. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, or more chromophores are attached to a primer. Insome cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50chromophores are attached to a primer. In some cases, one chromophore isattached to a primer. In some cases, one chromophore is attached at the5′ end of a primer. In some cases, a plurality of chromophores isattached to a primer with at least one chromophore attached at the 5′end of the primer.

In some cases, a plurality of fluorophores and quenchers is attached toa probe. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,or more fluorophores are attached to a probe. In some cases, about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more fluorophores areattached to a probe. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, or 50 fluorophores are attached to a probe. In somecases, one fluorophore is attached to a probe. In some cases, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more quenchers are attachedto a probe. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, or more quenchers are attached to a probe. In some cases, lessthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quenchers are attachedto on a probe. In some cases, one quencher is attached to a probe.

In some cases, a combination of fluorophores and quenchers are attachedto a probe. In some cases, the number of fluorophores and quenchers on aprobe is defined by a ratio. In some cases, the ratio of fluorophore toquencher is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80,90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some cases,the ratio of fluorophore to quencher is about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700,800, 900, or 1000. In some cases, the ratio of quencher to fluorophoreis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100,200, 300, 400, 500, 600, 700, 800, 900, or 1000. In some cases, theratio of quencher to fluorophore is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800,900, or 1000.

In some cases, a plurality of fluorophores and quenchers is attached toa primer. In some cases, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,or more fluorophores are attached to a primer. In some cases, about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more fluorophores areattached to a primer. In some cases, one fluorophore is attached to aprimer. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 fluorophores are attached to a primer. In some cases, onefluorophore is attached at the 5′ end of a primer. In some cases, aplurality of fluorophores is attached to a primer with at least onefluorophore attached to the 5′ end of the primer. In some cases, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more quenchers are attachedto a primer. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, or more quenchers are attached to a primer. In some cases, lessthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 quenchers are attachedto a primer. In some cases, one quencher is attached to a primer. Insome cases, one quencher is attached at the 5′ end of a primer. In somecases, a plurality of quenchers is attached to a primer with at leastone quencher attached at the 5′ end of the primer.

In some cases, a combination of fluorophores and quenchers are attachedto a primer. In some cases, the number of fluorophores and quenchers ona primer is defined by a ratio. In some cases, the ratio of fluorophoreto quencher is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60,70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. Insome cases, the ratio of quencher to fluorophore is about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500,600, 700, 800, 900, or 1000.

In some cases, multiple fluorophores are paired with one quencher. Insome cases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or morefluorophores are paired with one quencher. In some cases, about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more fluorophores are pairedwith one quencher. In some cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, or 50 fluorophores are paired with one quencher. In somecases, multiple fluorophore and quencher pairs are used. In some cases,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more fluorophore andquencher pairs are used. In some cases, about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, or more fluorophore and quencher pairs are used. Insome cases, less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, ormore fluorophore and quencher pairs are used. In some cases, 1fluorophore and quencher pair is used.

In some cases, a pair of primers comprises a first primer and a secondprimer. In some cases, multiple fluorophores are attached to a firstprimer and one quencher is attached to a second primer. In some cases,at least one fluorophore is attached to the 5′ end of the first primer.In some cases, the quencher is attached to the 5′ end of the secondprimer. In some cases, multiple fluorophores are attached to a secondprimer and one quencher is attached to a first primer. In some cases, atleast one fluorophore is attached to the 5′ end of the second primer. Insome cases, the quencher is attached to the 5′ end of the first primer.In some cases, a fluorophore and a quencher are not attached to the sameprimer.

The skilled artisan will realize that the advantages of the presentdisclosed probes or primers may be retained while modifying variousaspects of its structure. For example, but not by way of limitation, thenumber of donor/quencher pairs may be modified. The addition of moredonor/quencher pairs to the probe or primer is expected to increase theamount of total fluorescence observable prior to initiation ofamplification or polymerization reaction. There is no upper limit to thenumber of donor/quencher pairs that may be added to the probe or primer.In one example, the number of donor/quencher pairs is at least two. Inother examples, the detector contains at least three or moredonor/quencher pairs. In some examples, the detector may contain atleast 10, 20, 30, or 50 pairs, or it may contain hundreds ofdonor/quencher pairs, as needed to produce, for example, an optimalsignal-to-noise ratio and assay sensitivity.

In some cases, the methods provided in this disclosure may include theuse of fluorophore/quencher pair as a control. The controlfluorophore/quencher pair may be attached to one or more probe or primerpairs binding a positive control analyte, and each analyte to bedetected, in a sample. If the same sequence occurs in the positivecontrol analyte and each analyte to be detected, a single control primerpair may be used. If the same sequence does not occur in the positivecontrol analyte and each analyte to be detected, different primer pairsmay be used, but each primer pair may still be attached to the controlfluorophore/quencher pair.

For example, building on the methods described above, onefluorophore/quencher pair may be used to encode the presence of acontrol analyte that is always present in the sample. The controlanalyte may be added to the sample, or may be inherently present in thesample. Additional fluorophore/quencher pairs may be used to encode thepresence of additional analytes.

B. Signals

Disclosed herein is a method of utilizing the signal to identify thepresence or absence of an analyte. In some cases, the signal is anincrease in signal. In some cases, the signal is a decrease in signal.In some cases, a signature profile is generated based on the changes insignal at a specific distance. In some cases, once the length of ananalyte is known, additional information can be extrapolated. In somecases, additional information includes the molecular weight of ananalyte.

In some cases, a signal can be compared to a threshold value. Athreshold can be defined as the average standard deviation of a baseline(or background) signal multiplied by an adjustable factor. In somecases, during a PCR experiment, reaction and environmental conditionsrelated to each tube can influence fluorescence, and the fluorescencesignal may fluctuate over time creating a background signal or baselinesignal. This baseline signal can be determined, e.g, using the initialcycles of the PCR experiment used to detect an analyte. Alternatively,the background or baseline signal can be determined using a separateexperiment. A standard deviation calculated from the mean of thebaseline signal can then be used to establish a threshold. In somecases, the adjustable factor can be a factor of about 1, 1.1, 1.2, 1.3,1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100,or more. In some cases, the adjustable factor can be calculated prior tothe start of an experiment. In some cases, the adjustable factor canvary with the type of chromophore used. In some cases, the adjustablefactor can vary from experiment to experiment. In some cases, theadjustable factor can vary from instrument to instrument. In some cases,the adjustable factor is calculated for each instrument. In some cases,a threshold is established above the baseline signal. In some cases, athreshold is established below the baseline signal. In some cases, anobserved signal (e.g. the signal that is measured to detect an analyte)crosses the threshold level. In some cases, an observed signal does notcross the threshold level. In some cases, the observed signal thatcrosses the threshold indicates a presence of the product analyte. Insome cases, the observed signal that does not cross the thresholdindicates an absence of the product analyte.

In some cases, a reference signal or reference signal range isestablished which can be used, for example, as a control for any of themethods described herein. In some cases, the reference signal orreference signal range can be used to determine whether an amplificationor polymerization reaction described herein is performed successfully.For example, prior to initiating a PCR experiment involving the use of achromophore, a reference signal range for the chromophore can begenerated. The reference signal range can be generated by determiningthe values of two components, X and Y, such that the reference signalrange is X±Y. In some cases, the X component is the mean fluorescencesignal of the chromophore with respect to temperature and time, and theY component is the standard deviation of X. When used as a controlduring a PCR reaction, a signal from each denaturation step can becompared with the reference signal or reference signal range. A signaloutside of the reference signal range can indicate that the reaction hasfailed while a signal within the reference signal range can indicatethat the reaction has succeeded. In some cases, the reference signal orreference signal range can be used as a cycle-by-cycle control. In somecases, the reference signal or reference signal range can be used as aninternal control. In some cases, the method of calculating the referencesignal or reference signal range is the same as calculating the baselinesignal, e.g. establishing the mean of a fluorescence signal andcalculating its standard deviation.

In some cases, the denaturation signal is used for normalization duringan amplification or polymerization experiment. For example, when thedenaturation signal is within the reference signal range, thedenaturation signal can be used for normalization. During the course ofeach amplification or polymerization cycle, the denaturation signal canbe used to normalize the signal measured during the annealing step,thereby generating an internal normalization for each cycle, e.g.similar to the chopper stabilization where the signal can be reset to aparticular value or unit, for example, a value of 1. In some cases, thedenaturation signal is used for internal or self normalization. In somecases, the methods described herein use the denaturation signal of eachcycle for a cycle-by-cycle self-normalization.

In some cases, the annealing signal is used to determine the presence orabsence of an analyte. In some cases, when the annealing signals arecompared between consecutive steps using the cycle-by-cycleself-normalization method described previously, a relative change insignals can be determined. In some cases, the relative signal change canbe referred to as a relative quantitation of signals. In some cases, therelative quantitation of signals is used to determine the presence orabsence of an analyte. In some cases, the annealing signal is comparedto a standard to determine the presence of an analyte. In some cases,the standard is referred to as a standard ladder or a control signatureprofile, described elsewhere herein in this disclosure. In some cases,the comparison of the annealing signal to a standard ladder is referredto as a relative quantification of the analyte. In some cases, themethods described herein use a relative quantification method todetermine the presence or absence of an analyte.

In some cases, the methods presented in this disclosure may be used withany quantifiable signal. As described herein and elsewhere herein, acoding scheme may be utilized to indicate a multiplicity of signalsbased on color. In some cases, the coding scheme is equally applicableto any other method providing a quantifiable signal, including anelectrochemical signal and a chemiluminescent signal.

In some cases, if a fluorescent signal is employed, the number ofanalytes that can be encoded may be further expanded by utilizingadditional fluorophores. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, or more fluorophores may be used. In some cases, at least 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 fluorophores may be used.In some cases, fewer than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 fluorophores may be used.

Signals can be measured and compared at various points during adetection method described herein. For example, during an amplificationor polymerization reaction (e.g., a PCR reaction or process), pairwisesignals can be measured. In some cases, a signal can be measured: beforeannealing, during or after annealing of the primers to the template(e.g., analyte); before, during or after denaturing the double strandedtemplate; or before the annealing step or after the denaturing step. Asignal signature as described herein can be generated using thesemeasurements.

C. Signature Profiles

A signature profile typically comprises a plurality of signals. In somecases, a signal includes an electrochemical signal, a chemiluminescencesignal and a fluorescence signal. In some cases, a signature profilecontains a plurality of fluorescence signals. In some cases, a profilecurve is generated from the plurality of florescence signals. In somecases, a signature profile contains an initial fluorescence signal andan end-point fluorescence signal. In some cases, a signature profilecontains signals measured during the annealing step of an amplificationor polymerization reaction. In some cases, a fluorescence signal isinfluenced by external factors. In some cases, the external factorsinclude temperature, pH, organic and inorganic agents (e.g. salts, urea,DMSO) and addition or removal of chromophores.

In some cases, signature profiles are generated from different types ofdetection experiments. In some cases, a signature profile generated froma polynucleotide morphology study is referred to as a morphology curve.In some cases, a signature profile generated from a denaturation studyis referred to as a melt curve. In some cases, a signature profilegenerated from a persistence length study is referred to as a lengthcurve. In some cases, a signature profile generated from asingle-nucleotide polymorphism (SNP) study is referred to as a SNPcurve.

In some cases, the change in signal can be calculated as a percentage ofchange. In some cases, the percentage of signal change is 0.01, 0.1,0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70,75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900,1000, 10,000%. In some cases, the percentage of signal change is about0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55,60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 10,000%.

VII. Analytical Techniques and Instrumentation

The methods described in this disclosure are compatible with a varietyof amplification or polymerization methods, including polymerase chainreaction (PCR), ligase chain reaction (LCR), replicase-mediatedamplification, strand-displacement amplification (SDA), “rolling circle”types of amplification, and various transcription associatedamplification or polymerization methods. See, e.g., PCR amplification:U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; LCR amplification:U.S. Pat. No. 5,516,663 and EP 0320308 B1; replicase-mediatedamplification: U.S. Pat. No. 4,786,600; SDA amplification: U.S. Pat.Nos. 5,422,252 and 5,547,861; rolling circle types of amplification:U.S. Pat. Nos. 5,714,320 and 5,834,252; and transcription associatedamplification: U.S. Pat. Nos. 5,399,491, 5,554,516, 5,130,238,5,437,990, 4,868,105 and 5,124,246, PCT Pub. WO 1988/010315 A1, and USPub. 2006-0046265 A1, which are hereby incorporated by reference.

In some cases, the polymerase chain reaction (PCR) is a multiplex-PCR, avariable number of tandem repeats (VNTR) PCR, an asymmetric PCR, longPCR, a nested PCR, a hot-start PCR, a Touchdown PCR, an assembly PCR, acolony PCR, a quantitative PCR (qPCR), an end point PCR, a reversetranscriptase PCR, a digital PCR, or a droplet digital PCR. In somecases, the PCR process is a quantitative PCR process.

In some cases, the PCR amplification or polymerization step of thepresent disclosure can be performed by standard techniques well known inthe art (See, e.g., Sambrook, E. F. Fritsch, and T. Maniatis, MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarborLaboratory Press (1989); U.S. Pat. No. 4,683,202; and PCR Protocols: AGuide to Methods and Applications, Innis et al., eds., Academic Press,Inc., San Diego (1990) which are hereby incorporated by reference). PCRcycling conditions typically consist of an initial denaturation step,which can be performed by heating the PCR reaction mixture to atemperature ranging from about 80° C. to about 105° C. for times rangingfrom about 1 to about 10 min. I-eat denaturation is typically followedby a number of cycles, ranging from about 1 to about 80 cycles, eachcycle usually comprising an initial denaturation step, followed by aprimer annealing step and concluding with a primer extension step.Enzymatic extension of the primers by a nucleic acid polymerase, e.g.Taq polymerase, produces copies of the template (e.g., an analyte) thatcan be used as templates in subsequent cycles. In some cases, thedenaturation temperature is about 85° C. to about 100° C. In some cases,the denaturation temperature is 85° C., 86° C., 87° C., 88° C., 89° C.,90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C.,99° C., or 100° C. In some cases, the denaturation temperature is atleast 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C.,93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., or 100° C. Insome cases, the denaturation temperature is no more than 85° C., 86° C.,87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C.,96° C., 97° C., 98° C., 99° C., or 100° C. In some cases, the annealingtemperature is about 25° C. to about 80° C. In some cases, the annealingtemperature is 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C.,32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C.,41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C., 49° C.,50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C., 58° C.,59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C., 67° C.,68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C., 76° C.,77° C., 78° C., 79° C., or 80° C. In some cases, the annealingtemperature is at least 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.,31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C.,40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C.,76° C., 77° C., 78° C., 79° C., or 80° C. In some cases, the annealingtemperature is no more than 25° C., 26° C., 27° C., 28° C., 29° C., 30°C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39°C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48°C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57°C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66°C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75°C., 76° C., 77° C., 78° C., 79° C., or 80° C. In some cases, theextension temperature is 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.,31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C.,40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C.,76° C., 77° C., 78° C., 79° C., or 80° C. In some cases, the extensiontemperature is at least 25° C., 26° C., 27° C., 28° C., 29° C., 30° C.,31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C.,40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48° C.,49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57° C.,58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66° C.,67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75° C.,76° C., 77° C., 78° C., 79° C., or 80° C. In some cases, the extensiontemperature is no more than 25° C., 26° C., 27° C., 28° C., 29° C., 30°C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39°C., 40° C., 41° C., 42° C., 43° C., 44° C., 45° C., 46° C., 47° C., 48°C., 49° C., 50° C., 51° C., 52° C., 53° C., 54° C., 55° C., 56° C., 57°C., 58° C., 59° C., 60° C., 61° C., 62° C., 63° C., 64° C., 65° C., 66°C., 67° C., 68° C., 69° C., 70° C., 71° C., 72° C., 73° C., 74° C., 75°C., 76° C., 77° C., 78° C., 79° C., or 80° C. In some cases, the numberof cycles ranges from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75 to about 80 cycles.

The methods provided herein are suitable for use with a variety ofdetection methods. For example, the methods may be applied using ananalytical technique that measures a fluorescent signal. For example,many real-time PCR, quantitative PCR and droplet digital PCR instrumentscomprising an excitation light source that enable the detection offluorescent signals can be used. Therefore, the methods of the presentdisclosure can be readily applied using instruments widely used in theart.

VIII. Diseases

The methods described herein can be used, for example, to detect one ormore analytes associated with a disease or one or more geneticvariations (e.g., a SNP) associated with a disease. A disease is anabnormal condition of an organism. In some cases, the organism is amammal, such as a human, non-human primate, mouse, rat, rabbit, goat,dog, cat, or cow. In some cases, the mammal is a human. In some cases,the human is a patient or subject. In some cases, the disease is agenetic disorder, an autoimmune disease, a neurological disease, acardiovascular disease or a cancer.

A genetic disorder is a disease caused by one or more abnormalities inthe genome. Exemplary genetic disorders include 22q11.2 deletionsyndrome, Acrocephaly, Acute cerebral Gaucher's disease, Adrenal glanddisorders, Adrenogenital syndrome, Alzheimer's disease, Amelogenesisimperfect, androgen insensitivity syndrome, anemia, Angelman syndrome,Apert syndrome, ataxia telangiectasia, Canavan disease,Charcot-Marie-Tooth disease, Color blindness, Cri du chat, Cysticfibrosis, Down syndrome, Duchenne muscular dystrophy, Haemochromatosis,Haemophilia, Klinefelter syndrome, Neurofibromatosis, Phenylketonuria,Polycystic kidney disease, Prader-Willi syndrome, Sickle-cell disease,Tay-Sachs disease and Turner syndrome.

An autoimmune disease is a disease caused when the immune systemmistakenly attacks and destroys healthy body tissue. Exemplaryautoimmune diseases include lopecia areata, autoimmune hemolytic anemia,autoimmune hepatitis, dermatomyositis, diabetes (type 1), several formsof juvenile idiopathic arthritis, glomerulonephritis, Graves' disease,Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myastheniagravis, several forms of myocarditis, multiple sclerosis,pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa,polymyositis, primary biliary cirrhosis, psoriasis, rheumatoidarthritis, scleroderma/systemic sclerosis, Sjögren's syndrome, systemiclupus erythematosus, several forms of thyroiditis, several forms ofuveitis, vitiligo, and granulomatosis with polyangiitis (Wegener's).

Exemplary neurological diseases include attention deficit hyperactivitydisorder (ADHD), ALS, Alzheimer's disease, bipolar disorder, Bell'spalsy, birth defects of the brain and spinal cord, cerebral palsy,chronic fatigue syndrome, dyslexia, epilepsy, Guillain-Barré syndrome,multiple sclerosis, muscular dystrophy, neuropathy, neuromuscular andrelated diseases, Parkinson's disease, schizophrenia, scoliosis andspinal deformity.

Exemplary cardiovascular disease include acute myocardial infarction,angina, arrhythmia, atherosclerosis, cardiomegaly, cardiomyopathy,carotid artery disease, congenital heart disease, congestive heartfailure, coronary artery disease, endocarditis, fluid around the heart,hypertension, infective endocarditis, mitral valve prolapsed, peripheralartery disease, stroke, and valvular heart disease.

Cancer is characterized by an abnormal growth of cells. Exemplary cancerinclude bladder, brain, breast, bone, cervical, colon, esophageal,kidney, liver, lung, ovarian, pancreatic, proximal or distal bile duct,prostate, skin, stomach, thyroid, and uterine cancer.

In some cases, the presence of an analyte or a genetic variation in ananalyte (e.g., a SNP) can serve as a disease marker. In some cases, themethod disclosed herein can be used to detect a disease marker. In somecases, the method disclosed herein can be applicable in determining thepresence or absence or the type of diseases affecting a patient. Forexample, FIG. 3 illustrates an overview of a method of providing atreatment in conjunction with a detection method described herein. 601illustrates a clinician preparing to take a sample from a patient. Insome cases, the sample can be a blood sample. In some cases, the samplecan be a tissue sample. 602 illustrates a sample diluted into threeconcentrations (e.g. a separate concentration in each tube). 603indicates an amplification or polymerization step (e.g., PCR). 604illustrates products of the amplification or polymerization step. 605depicts a clinician returning the results of an analysis to a patient.

IX. Compositions and Kits

This disclosure also provides compositions and kits for use with themethods described herein. The compositions may comprise any component,reaction mixture and/or intermediate described herein, as well as anycombination thereof. For example, the disclosure provides detectionreagents for use with the methods provided herein. Any suitabledetection reagents may be provided, including a primer pair attached totwo different chromophores (e.g., a fluorophore and a quencher), asdescribed elsewhere in the specification.

In some cases, compositions comprise a first and a second primer orprobe for the detection of at least one analyte wherein the primers areattached to either a fluorophore or a quencher at the 5′ end. In somecases, compositions comprise primers attached at the 5′ end with eithera fluorophore or a quencher for the detection of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, or 10000 analytes.In some cases, compositions comprise primers attached to multipledifferent fluorophores or quenchers wherein at least one fluorophore orquencher is at the 5′ end for the detection of at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 100, 500, 1000, 5000, or 10000 analytes.In some cases the compositions comprise multiple pairs of first andsecond primers, wherein each pair of first and second primers compriseeither a fluorophore or a quencher at the 5′ end. In some cases eachpair of first and second primers comprise a different fluorophore andquencher from the remaining set of primers. In some cases thecompositions comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 100, 500, 1000, 5000, or 10000 pairs of first and secondprimers.

The present disclosure also provides kits for carrying out the methodsof the invention. Accordingly, a variety of kits are provided insuitable packaging. The kits may be used for any one or more of the usesdescribed herein, and, accordingly, may contain instructions fordetecting the presence or absence of each analyte or a plurality ofanalytes. A kit may be a diagnostic kit, for example, a diagnostic kitsuitable for the detection of one or more analytes, including theanalytes recited herein. A kit may contain any of the compositionsprovided in this disclosure, including those recited above.

X. Services

The methods provided herein may also be performed as a service. Forexample, a service provider may obtain the identity of a plurality ofanalytes that a customer wishes to analyze. The service provider maythen encode each analyte to be detected by any of the methods describedherein and provide appropriate reagents to the customer for the assay.The customer may perform the assay and provide the results to theservice provider for decoding. The service provider may then provide thedecoded results to the customer. The customer may also encode analytes,generate probes, and/or decode results by interacting with softwareinstalled locally (at the customer's location) or remotely (e.g., on aserver reachable through a network). Exemplary customers includeclinical laboratories, physicians, manufacturers of food and consumerproducts, industrial manufacturers (e.g., petroleum companies) and thelike. A customer or party may be any suitable customer or party with aneed or desire to use the methods, systems, compositions, and kits ofthe invention.

A. Server

The methods provided herein may be processed on a server or a computerserver (FIG. 4). The server 1101 includes a central processing unit(CPU, also “processor”) 1105 which can be a single core processor, amulti core processor, or plurality of processors for parallelprocessing. A processor used as part of a control assembly may be amicroprocessor. The server 1101 also includes memory 1110 (e.g. randomaccess memory, read-only memory, flash memory); electronic storage unit1115 (e.g. hard disk); communications interface 1120 (e.g. networkadaptor) for communicating with one or more other systems; andperipheral devices 1125 which may include cache, other memory, datastorage, and/or electronic display adaptors. The memory 1110, storageunit 1115, interface 1120, and peripheral devices 1125 are incommunication with the processor 1105 through a communications bus(solid lines), such as a motherboard. The storage unit 1115 can be adata storage unit for storing data. The server 1101 is operativelycoupled to a computer network (“network”) 1130 with the aid of thecommunications interface 1120. A processor with the aid of additionalhardware may also be operatively coupled to a network. The network 1130can be the Internet, an intranet and/or an extranet, an intranet and/orextranet that is in communication with the Internet, a telecommunicationor data network. The network 1130 in some cases, with the aid of theserver 1101, can implement a peer-to-peer network, which may enabledevices coupled to the server 1101 to behave as a client or a server. Ingeneral, the server may be capable of transmitting and receivingcomputer-readable instructions (e.g., device/system operation protocolsor parameters) or data (e.g., sensor measurements, raw data obtainedfrom detecting nucleic acids, analysis of raw data obtained fromdetecting nucleic acids, interpretation of raw data obtained fromdetecting nucleic acids, etc.) via electronic signals transportedthrough the network 1130. Moreover, a network may be used, for example,to transmit or receive data across an international border.

The server 1101 may be in communication with one or more output devices1135 such as a display or printer, and/or with one or more input devices1140 such as, for example, a keyboard, mouse, or joystick. The displaymay be a touch screen display, in which case it may function as both adisplay device and an input device. Different and/or additional inputdevices may be present such an enunciator, a speaker, or a microphone.The server may use any one of a variety of operating systems, such asfor example, any one of several versions of Windows, or of MacOS, or ofUnix, or of Linux.

The storage unit 1115 can store files or data associated with theoperation of a device or method described herein.

The server can communicate with one or more remote computer systemsthrough the network 1130. The one or more remote computer systems maybe, for example, personal computers, laptops, tablets, telephones, Smartphones, or personal digital assistants.

In some situations a control assembly includes a single server 1101. Inother situations, the system includes multiple servers in communicationwith one another through an intranet, extranet and/or the Internet.

The server 1101 can be adapted to store device operation parameters,protocols, methods described herein, and other information of potentialrelevance. Such information can be stored on the storage unit 1115 orthe server 1101 and such data can be transmitted through a network.

EXAMPLES Example 1: DNA Sequences, Primers and Probes

Synthetic nucleic acid analytes from the Human Immunodeficiency Virus 1(HIV-1) poly protease gene were chosen for exemplary detection studiesusing an amplification or polymerization technique. Templates from thisgene, of lengths varying from 40 bp-80 bp, were synthesized.

Seven nucleic acid analytes from organisms of clinical relevance werechosen for exemplary detection studies using a multiplex PCR technique.These analytes include Influenza type A (InfA), influenza type B (InfB),respiratory syncytial virus type A (RsvA), respiratory syncytial virustype B (RsvB), human rhinovirus (Hrv), and human metapneumovirus (Hmpv).An additional analyte for human parainfluenza virus type 3 (PIV-3) wasincluded to run as an orthogonal control. Multiplex analytes were codedusing a binary scheme described elsewhere herein in this disclosure.

All oligonucleotides were synthesized by Integrated DNA Technologies(Coralville, Iowa). Diagnostic sequences were input into IDT'sOligoAnalyzer 3.1 tool. Probes and primer pairs for each analyte werechosen, from the OligoAnalyzer's set of generated sequences, to minimizehomology with un-intended analytes, probes and primers. Forward primersfor all analytes were synthesized with a fluorophore at the 5′ end,while the reverse primes for all analytes were synthesized with aquencher at the 5′ end. Sequence information is tabulated in Tables 1 to8. Nucleic acid products were synthesized and lyophilized by IDT. Allnucleic acid analytes were reconstituted in TE buffer, ph7 (LifeTechnologies, Carlsbad, Calif.). Dilutions were done using UltraPureRNAse-free Water (Life Technologies, Carlsbad, Calif.).

Example 2: Primer Design

Specific primers and probes were designed using conserved regions of theanalyte genes. Primers for Influenza A and B were designed in thenucleoprotein genes; Primers for RSV A and RSV B were designed in theconserved Fusion protein gene; Primers for HRV were designed in the5′-UTR region; Primers for HMPV were designed in the Fusion proteingene; Primers for human PIV-3 were designed in conserved regions of thehaemagglutinin genes.

Established GenBank Accession nos. were used for Influenza A, InfluenzaB, HRV, HMPV, and PIV-3. For RSV A and RSV B, primers used in previousstudies (Jansen et al. “Development and evaluation of a four-tube realtime multiplex PCR assay covering fourteen respiratory viruses, andcomparison to its corresponding single target counterparts.” Journal ofClinical Virology 51.3 (2011): 179-185; and Chun et al. “Dual primingoligonucleotide system for the multiplex detection of respiratoryviruses and SNP genotyping of CYP2C19 gene.” Nucleic Acids Research 35.6(2007): e40) were inputted into BLAST and Accession nos. correspondingto high homology with complete coding sequence information were chosen.Accession nos. for virus sequences used in primer design can be found inTables 2-8. Sequences were uploaded into Primer Quest software(Integrated DNA Technologies, Coralville, Iowa) with parameters set totake into account primer size, amplicon size, G-C %, similarity ofreaction kinetics. A BLAST search was performed to check the specificityof the sequences of the primers and probes. Chosen primer pairs wereentered into OligoAnalyzer 3.1 and analyzed for self-dimerization andhetero-dimerization. Primer sequences and their properties can be foundin Tables 2-8. Sequence identifier numbers can be found in Tables 1-8.

TABLE 1 HIV-1 Poly Protease Sequence Information Sequence SEQ ID NO.Information HIV-1 40mer, 60mer, and 80mer SourceHIV-1 Reference Sequence, Los Alamos National Laboratory SEQ ID NO: 140Mer 5′-GGA AGC TCT ATT AGA TAC AGA CAC CTG TemplateTCA ACA TAA TTG G-3′ SEQ ID NO: 2 60Mer5′-GGA AGC TCT ATT AGA TAC AGA TGA TAC TemplateAGT ATT AGA AGA AAC ACC TGT CAA CAT AAT TGG-3′ SEQ ID NO: 3 80Mer5′-GGA AGC TCT ATT AGA TAC AGA TGA TAC TemplateAGT ATT AGA AGA ATA GAG TTT GCC AGG AAGATG ACA CCT GTC AAC ATA ATT GG-3′ SEQ ID NO: 4 FWD Primer5′-/5Cy3/GG AAG CTC TAT TAG ATA CAG-3′ SEQ ID NO: 5 RWD Primer5′-/5IABkFQ/CC AAT TAT GTT GAC AGG TGT-3′

TABLE 2 Influenza type A sequence information Sequence SEQ ID NO.Information Influenza A Source GenBank Accession #M23976.1 Influenza ASEQ ID NO: 6 85Mer 5′-GTA GGG ATA GAC CCT TTC AAA CTG CTT TemplateCAA AAC AGC CAA GTA TAC AGC CTA ATC AGACCG AAT GAG AAT CCA GCA CAC AAG AGT C-3′ SEQ ID NO: 7 FWD Primer5′-GTA GGG ATA GAC CCT TTC AAA CTG-3′ SEQ ID NO: 8 RWD Primer5′-GAC TCT TGT GTG CTG GAT TCT C-3′ SEQ ID NO: 9 TaqMan5′-/56-FAM/AG CCA AGT ATA CAG CCT AAT Probe CAG ACC GA/3BHQ_1/-3′SEQ ID NO: 10 FWD qPrimer 5′-/5Cy3/GT AGG GAT AGA CCC TTT CAA ACT G-3′SEQ ID NO: 11 RWD qPrimer 5′-/5IABkFQ/GA CTC TTG TGT GCT GGA TTC TC-3′SEQ ID NO: 12 FWD Mistake 5′-/5Cy3/GT AGG GAG AGA CCC TTT CAA ACTqPrimer G-3′

TABLE 3 Influenza type B sequence information Sequence SEQ ID NO.Information Influenza B Source GenBank Accession # AB036876 Influenza BSEQ ID NO: 13 81Mer 5′-GTG CTT CCC ATA AGC ATT TAC GCC AAA TemplateATA CCT CAA CTA GGG TTC AAC GTT GAA GAGTAC TCT ATG GTT GGG TAT GAA GCC-3′ SEQ ID NO: 14 FWD Primer5′-GTG CTT CCC ATA AGC ATT TAC G-3′ SEQ ID NO: 15 RWD Primer5′-GGC TTC ATA CCC AAC CAT AGA G-3′ SEQ ID NO: 16 TaqMan5′-/56-FAM/CC TCA ACT AGG GTT CAA CGT Probe TGA AGA GT/3BHQ_1/-3′SEQ ID NO: 17 FWD qPrimer 5′-/5Cy3/GT GCT TCC CAT AAG CAT TTA CG-3′SEQ ID NO: 18 RWD qPrimer 5′-/5IABkFQ/GG CTT CAT ACC CAA CCA TAG AG-3′

TABLE 4 Respiratory syncytial virus type A sequence information SequenceSEQ ID NO. Information Respiratory syncytial virus type A SourceGenBank Accession # |JX627336.1|:5726-7450 Human respiratory syncytial virusstrain RSVA/GN435/11, complete genome SEQ ID NO: 19 85Mer5′-GTT GGA AAC TAC ACA CAT CTC CTC TAT TemplateGTA CAA CCA ACA CAA AGG AAG GAT CCA ACATCT GCT TAA CAA GAA CCG ACA GAG GAT G-3′ SEQ ID NO: 20 FWD Primer5′-GTT GGA AAC TAC ACA CAT CTC CTC-3′ SEQ ID NO: 21 RWD Primer5′-CAT CCT CTG TCG GTT CTT GTT AG-3′ SEQ ID NO: 22 TaqMan5′-/56-FAM/CC AAC ACA AAG GAA GGA TCC Probe AAC ATC TG/3BHQ_1/-3′SEQ ID NO: 23 FWD qPrimer 5′-/5Cy3/GT TGG AAA CTA CAC ACA TCT CCT C-3′SEQ ID NO: 24 RWD qPrimer 5′-/5IABkFQ/CA TCC TCT GTC GGT TCT TGTTAA G-3′

TABLE 5 Respiratory syncytial virus type B sequence information SequenceSEQ ID NO. Information Respiratory syncytial virus type B SourceGenBank Accession # JX682822.1 SEQ ID NO: 25 81Mer5′-CCT CAC CTC AAG TCA GAA CAT AAC TGA TemplateGGA GTT TTA CCA ATC GAC ATG TAG TGC AGTTAG CAG AGG TTA CTT GAG TGC TTT-3′ SEQ ID NO: 26 FWD Primer5′-CCT CAC CTC AAG TCA GAA CAT AC-3′ SEQ ID NO: 27 RWD Primer5′-AAA GCA CTC AAG TAA CCT CTG C-3′ SEQ ID NO: 28 TaqMan5′-/56-FAM/AC CAA TCG ACA TGT AGT GCA Probe GT/3BHQ_1/-3′ SEQ ID NO: 29FWD qPrimer 5′-/5Cy3/CC TCA CCT CAA GTC AGA ACA TAA C-3′ SEQ ID NO: 30RWD qPrimer 5′-/5IABkFQ/AA AGC ACT CAA GTA ACC TCT GC-3′

TABLE 6 Human rhinovirus sequence information Sequence SEQ ID NO.Information Human rhinovirus Source GenBank Accession # AF108174.1SEQ ID NO: 31 81Mer 5′-ACA ATG GAC AG GTG TGA AGA GCC CCG TemplateTGT GCT CGC TTT GAG TCC TCC GGC CCC TGAATG TGG CTA ACC TTA ACC CTG CAG CTA G-3′ SEQ ID NO: 32 FWD Primer5′-ACA ATG GAC AAG GTG TGA AGA G-3′ SEQ ID NO: 33 RWD Primer5′-CTA GCT GCA GGG TTA AGG TTA G-3′ SEQ ID NO: 34 TaqMan5′-/56-FAM/TG TGC TCG CTT TGA GTC CTC Probe CG/3BHQ_1/-3′ SEQ ID NO: 35FWD qPrimer 5′-/5Cy3/AC AAT GGA CAA GGT GTG AAG AG-3′ SEQ ID NO: 36RWD qPrimer 5′-/5IABkFQ/CT AGC TGC AGG GTT AAG GTT AG-3′

TABLE 7 Human metapneumovirus sequence information Sequence SEQ ID NO.Information Human metapneumovirus Source GenBank Accession #|AF371337.2|:3052- 4671 Human metapneumovirus isolate 00-1,complete genome) SEQ ID NO: 37 88Mer5′-GAG AGC ATT GAG AAC AGT CAG GCC TTG TemplateGTG GAT CAA TCA AAC AGA ATC CTA AGC AGTGCA GAG AAA GGA AAC ACT GGC TTC ATC ATT G-3′ SEQ ID NO: 38 FWD Primer5′-GAG AGC ATT GAG AAC AGT CAG G-3′ SEQ ID NO: 39 RWD Primer5′-CAA TGA TGA AGC CAG TGT TTC C-3′ SEQ ID NO: 40 TaqMan5′-/56-FAM/AC AGA ATC CTA AGC AGT GCA Probe GAG A/3BHQ_1/-3′SEQ ID NO: 41 FWD qPrimer 5′-/5Cy3/GA GAG CAT TGA GAA CAG TCA GG-3′SEQ ID NO: 42 RWD qPrimer 5′-/5IABkFQ/CA ATG ATG AAG CCA GTG TTT CC-3′

TABLE 8 Parainfluenza virus type 3 sequence information SequenceSEQ ID NO. Information Parainfluenza virus type 3 SourceGenBank Accession # gi|403376|emb|Z26523.1| Humanparainfluenza virus type 3 HN gene for hemagglutinin-neuraminidase)SEQ ID NO: 43 86Mer 5′-TCG AGA GTG AAC CCA GTC ATA ACT TAC TemplateTCA ACA GCA ACC GAA AGA GTA AAC GAG CTGGCC ATC CGA AAC AGA ACA CTC TCA GCT GG-3′ SEQ ID NO: 44 FWD Primer5′-TCG AGA GTG AAC CCA GTC ATA A-3′ SEQ ID NO: 45 RWD Primer5′-CCA GCT GAG AGT GTT CTG TTT C-3′ SEQ ID NO: 46 TaqMan5′-/56-FAM/CC GAA AGA GTA AAC GAG CTG Probe 1 GCC A/3BHQ_1/-3′SEQ ID NO: 47 TaqMan 5′-/5Cy3/CC GAA AGA GTA AAC GAG CTG GCC Probe 2A/3BH_Q2/-3′ SEQ ID NO: 48 TaqMan 5′-/56-ROXN/CC GAA AGA GTA AAC GAG CTGProbe 3 GCC A/3BH_Q2/-3′ SEQ ID NO: 49 TaqMan5′-/5Cy5/CC GAA AGA GTA AAC GAG CTG GCC Probe 4 A/3IAbRQSp/-3′

Example 3: Polymerase Chain Reactions

PCR reactions were performed on a Roche 480 LightCycler instrument(Roche Applied Science, Penzberg, Germany). The PCR cycling reaction wasrun for 45 cycles, with a 60 sec hot-start at 95° C. The cyclingconditions were: denaturation for 45 sec at 95° C., annealing for 120sec at 45° C., and extension for 120 sec at 58° C. Each experiment wasrun in sextuplicate, with a reaction volume of 15 μL. Fluorescencemeasurements in 523 nm-568 nm (Cy3) and 615 nm-670 nm (Cy5) were takenat the end of annealing for every cycle. The change in fluorescenceintensity between the first and last measurements (the quenched signal)was measured for each experiment.

Positive control experiments were performed to determine base-linequenching levels for each analyte and set of primers. Only analytes withtheir associated probes were cycled in experiments 1-10, and tabulatedin Tables 9-18.

In each experiment, the uncertainty in the cycling data was determinedby the spread of values in the last five cycles of the particularamplification reaction. This uncertainty did not scale with the value ofthe total signal, which implied that the source of uncertainty wasinstrumental rather than experimental. A 1× fluorophore (200 nM)baseline was determined by statistical analysis of a set of data on 200nM concentration. The expected multiplicative signal levels weredetermined by multiplying this baseline by the multiplicity. The changein fluorescence intensity was used to assemble the expected signallevels in the chromatograms depicted in FIGS. 5-9.

Example 4: Detection of HIV TPP Analytes

FIG. 5 illustrates the successful detection of HIV TPP analytes usingthe analytes, primers and methods described in Examples 1-3. Initialexperiments showed the quantitative PCR (qPCR) detection of 80 bp HIVTPP analytes at varying concentrations from 10 nM to 10 pM (A-D). Allsuccessful detection curves exhibited the inverse-sigmoidalcharacteristic. FIG. 6 indicates the detection assay can be used todetect low concentrations of HIV TPP. The experiments demonstrated thedetection assay was able to detect between 1 pM and 100 fM (FIG. 6A andFIG. 6B) of HIV TPP. FIG. 7 illustrates the length dependence ofquenching. Experiments showed that the extent of quenching is stronglycorrelated to the separation length between a quencher and a fluorophore(FIG. 7A-7C). Analytes were varied in length from 40 bp to 120 bp for asingle set of primers.

Example 5: Detection of Single Nucleotide Polymorphisms

FIG. 8 illustrates the detection of a single nucleotide polymorphism.Signal level changed significantly (approximately a 35% reduction insignal) when a mis-priming event occurred (FIG. 8A and FIG. 8B). Theobserved effect suggests the mechanism of signal change involved theelectron transport between chromophores of the labeled analyte.

Example 6: Multiplex Detection Using Binary Coding of Analytes

FIG. 9 illustrates the binary coding of the analytes. Analytes in a3-plex CY3 assay were coded using binary spaced primer concentrations. ATaqMan positive control sequence was amplified in every reaction toconfirm that the PCR completed successfully.

Example 7: Tagged Primer PCR

This example describes a method of directly detecting a DNA analyte in asample by incorporating chromophores onto primers and performing a PCRreaction. This method directly detects a PCR product, and does notrequire a secondary detection technique, such as a gel electrophoresis.For instance, an exemplary analyte is illustrated in FIG. 10A (SEQ IDNO: 50). A forward (FWD) (SEQ ID NO: 51) and reverse or rewind (RWD)(SEQ ID NO: 52) primers are designed such that a quencher andfluorophore are ligated to the 5′ of each primer, respectively (FIG.10B). The primers are selected in appropriate concentrations to limitunbound quenching. Before the first cycle of the PCR reaction is run,the total fluorescence intensity is 10. During the first cycle of thePCR, this intensity level is typically unchanged, as there is no DNAanalyte present that incorporates both a fluorophore and a quencher(FIG. 10C; SEQ ID NO: 50-SEQ ID NO: 52). A decrease in signal isobserved upon completion of a second PCR cycle (FIGS. 10D and 10E; SEQID NO: 50-SEQ ID NO: 52). As the cycles progress, on average, the totalfluorescent signal decreases by a factor of 2 per cycle. Therefore, thefluorescent intensity at the end of cycle two is approximately 10/2because the fluorophore on the anti-sense strand is quenched by thequencher on the sense strand. This attenuation of the fluorescent signalcontinues until the supply of primers for that analyte is exhausted. Inthis way, a “dark-field” measurement for the DNA analyte is obtained bylooking at the intensity of fluorescence after each cycle.

Example 8: Multiplex Detection Using Various Chromophore Combinations

This example describes a technique for distinguishing multiple DNAanalytes in a single PCR reaction using various chromophore combinationsfor each pair of analyte-specific primers. Color multiplexing differentstrands as illustrated in FIG. 11 is used to distinguish betweenmultiple analytes. Dark-field curves are generated after performing PCR.From these dark-field curves, the initial concentrations areinterpolated.

Example 9: Multiplex Detection Using a Single Chromophore Combination

This example describes a technique for distinguishing multiple DNAanalytes of varying lengths in a single PCR reaction using the samechromophore combination for each pair of analyte-specific primers.Analytes of differing lengths (e.g., 40 bp, 60 bp, and 80 bp) aredetected using a single fluorophore/quencher combination. Longeranalytes have a greater separation between the quencher and thefluorophore, and thus exhibit different end point fluorescence levels.In superposition, different combinations of analytes code for differentfluorescence levels. Further discrimination of nucleic acid analytes isperformed using a melt curve analysis. Lower molecular weight analytesdenature at lower temperatures. By measuring fluorescence as a functionof increasing temperature, multiple analytes in a single color channelare quantified.

Example 10: Detection of Morphology

This example describes the characterization of the morphology of ananalyte (e.g., a DNA analyte) using multiple chromophores. The DNAanalyte illustrated in FIG. 12 (SEQ ID NO: 50) has a particularmorphology at a given temperature. This morphology results in afluorescence signature. Changing the temperature results in a differentmorphology of the DNA analyte, which in turn results in a differentfluorescence signature (FIG. 13). The signatures for the morphology ofanalytes (such as the DNA molecule shown in FIG. 12) are obtained byramping of the temperature and determining fluorescent signatures. Bysuperimposing these signatures, a three dimensional folding model forthe DNA molecule is obtained.

While preferred disclosures of the present disclosure have been shownand described herein, it will be obvious to those skilled in the artthat such disclosures are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the present disclosure. It should beunderstood that various alternatives to the disclosures described hereinmay be employed in practicing the invention. It is intended that thefollowing claims define the scope of the invention and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

TABLE 9 Experiment 01 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 40mer 12 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 10 Experiment 02 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 60mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 11 Experiment 03 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 80mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 12 Experiment 04 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 45mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 13 Experiment 05 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 50mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 14 Experiment 06 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 55mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 15 Experiment 07 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 100mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 16 Experiment 08 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 70 μL Taq 5× Master Mix — 25 μL Templates ConcentrationVolume Added Poly Protease 120mer 10 nM  5 μL Primers ConcentrationVolume Added Poly Protease FWD qPrimer  1 μM  5 μL Poly Protease RWDqPrimer  1 μM  5 μL

TABLE 17 Experiment 09 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 105 μL Taq 5× Master Mix —  75 μL TemplatesConcentration Volume Added Influenza A 85mer  10 nM  5 μL PrimersConcentration Volume Added Influenza A FWD qPrimer 300 nM  5 μLInfluenza A RWD qPrimer 300 nM  5 μL

TABLE 18 Experiment 10 Cocktail Reagents Concentration Volume AddedUltraPure H₂O — 105 μL Taq 5× Master Mix —  75 μL TemplatesConcentration Volume Added Influenza B 81mer  10 nM  5 μL PrimersConcentration Volume Added Influenza B FWD qPrimer 300 nM  5 μLInfluenza B RWD qPrimer 300 nM  5 μL

What is claimed is:
 1. A method of detecting at least one geneticvariation in a polynucleotide analyte in a sample, comprising: a)combining the sample comprising the polynucleotide analyte with a firstprimer and a second primer, wherein a fluorophore is attached to thefirst primer, a quencher is attached to the second primer, the firstprimer and the second primer are specific for the polynucleotide analyteand the fluorophore is different from the quencher; b) measuring a firstsignal generated by the fluorophore and quencher; c) performing at leastone polymerization reaction with the first primer and the second primerusing the polynucleotide analyte as a template; d) measuring a secondsignal generated by the fluorophore and quencher; e) comparing the firstand second signals to determine a change between the first and secondsignals; and f) determining the presence or absence of the at least onegenetic variation based i) on the change in signal as determined in stepe); and ii) by further comparing said change to the change in signalobserved for an analyte without the at least one genetic variation.
 2. Amethod for detecting at least one genetic variation in a polynucleotideanalyte in a sample, comprising: a) combining the sample comprising thepolynucleotide analyte with a first primer and a second primer, whereina fluorophore is attached to the first primer, a quencher is attached tothe second primer, the first primer and the second primer are specificfor the polynucleotide analyte, and the fluorophore is different fromthe quencher; b) measuring a first signal generated by the fluorophoreand quencher; c) after measuring the first signal, performing at leastone polymerization reaction with the first primer and the second primerusing the polynucleotide analyte as a template; d) after performing theat least one polymerization reaction, measuring a second signalgenerated by the fluorophore and quencher; e) comparing the first andsecond signals to determine a first change in signal; and f) determiningthe presence or absence of the at least one genetic variation based i)on the first change in signal as determined in step e); and ii) bycomparing the first change in signal to a second change in signalobserved from performing steps a) through e) for a correspondingpolynucleotide analyte without the at least one genetic variation. 3.The method of claim 2, wherein the at least one genetic variationcomprises a single-nucleotide polymorphism (SNP).
 4. The method of claim2, wherein the fluorophore is attached to the 5′ end of the firstprimer.
 5. The method of claim 2, wherein the fluorophore is 6-FAM(Fluorescein), 6-FAM (NHS Ester), Fluorescein dT, HEX, JOE (NHS Ester),MAX, TET, ROX, TAMRA, TARMA (NHS Ester), TEX 615, ATTO 488, ATTO 532,ATTO 550, ATTO 565, ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563,TYE 665 or TYE
 705. 6. The method of claim 2, wherein the quencher isattached to the 5′ end of the second primer.
 7. The method of claim 2,wherein the quencher is Iowa Black FG, Iowa Black RG, BHQI, BHQ2 orBHQ3.
 8. The method of claim 3, wherein the first primer is configuredto hybridize to a region of the polynucleotide analyte encoding the SNP.9. The method of claim 3, wherein the second primer is configured tohybridize to a region of the polynucleotide analyte encoding the SNP.10. The method of claim 2, wherein the polynucleotide analyte is a DNApolynucleotide analyte or an RNA polynucleotideanalyte.
 11. The methodof claim 2, wherein the polynucleotide analyte is from about 10 to about500 nucleotides in length.
 12. The method of claim 2, wherein the atleast one genetic variation is present when the first change in signalis less than the second change in signal.
 13. The method of claim 2,wherein the first and second changes in signal are distinct for UU, UT,UG, UC, UA, AA, TT, GG, CC, AG, AC, TG, and TC.
 14. The method of claim2, wherein a decrease in fluorescent intensity is at least, or about, a30% decrease in signal.
 15. The method of claim 2, wherein the first andsecond primers are configured to amplify the polynucleotide analytehaving the at least one genetic variation and the correspondingpolynucleotide analyte lacking the at least one generic variation byperforming at least one polymerase chain reaction (PCR) reaction withthe first primer and the second primer, wherein the PCR reaction is anend-point polymerase chain reaction process, a real-time polymerasechain reaction process, a digital polymerase chain reaction process, adroplet digital polymerase chain reaction process, or a quantitativepolymerase chain reaction process.
 16. The method of claim 15, whereinthe PCR reaction is a quantitative PCR process.
 17. The method of claim2, wherein the concentration of the polynucleotide analyte is from about10 μM to about 10 aM.
 18. The method of claim 2, wherein the firstprimer is configured to encode a region on the polynucleotide analyteless than 500 base pairs away from a region encoded by the secondprimer.
 19. The method of claim 3, wherein the SNP is associated with adisease, optionally a genetic disorder, an autoimmune disease, aneurological disease, a cardiovascular disease, or a cancer.
 20. Themethod of claim 2, wherein the first primer is configured to hybridizeto the region of the polynucleotide analyte encoding the at least onegenetic variation, wherein when the first primer is hybridized to theregion of the polynucleotide analyte encoding the at least one geneticvariation, the fluorophore is configured to attach to a portion of thefirst primer that is 5′ to the site of the at least one geneticvariation.
 21. The method of claim 2, wherein the second primer isconfigured to hybridize to the region of the polynucleotide analyteencoding the at least one genetic variation, wherein when the secondprimer is hybridized to the region of the polynucleotide analyteencoding the at least one genetic variation, the quencher is configuredto attach to a portion of the second primer that is 5′ to the site ofthe at least one genetic variation.
 22. The method of claim 2, whereinthe at least one genetic variation comprises a deletion, an insertion, apoint mutation, a base-pair substitution, or a variation in the numberof multiple nucleotide repetition.
 23. The method of claim 2, whereinthe corresponding polynucleotide analyte is a wild-type analyte.